This study aimed to determine the factors which regulate soil microbial community organisation and function in temperate upland grassland ecosystems. Soil microbial biomass (Cmic), activity (respiration and potential carbon utilisation) and community structure (phospholipid fatty acid (PLFA) analysis, culturing and community level physiological profiles (CLPP) (Biolog®)) were measured across a gradient of three upland grassland types; Festuca–Agrostis–Galium grassland (unimproved grassland, National Vegetation Classification (NVC) — U4a); Festuca–Agrostis–Galium grassland, Holcus–Trifolium sub-community (semi-improved grassland, NVC — U4b); Lolium–Cynosurus grassland (improved grassland, NVC — MG6) at three sites in different biogeographic areas of the UK over a period of 1 year. Variation in Cmic was mainly due to grassland type and site (accounting for 55% variance, v, in the data). Cmic was significantly (P<0.001) high in the unimproved grassland at Torridon (237.4 g C m−2 cf. 81.2 g C m−2 in semi- and 63.8 g C m−2 in improved grasslands) and Sourhope (114.6 g C m−2 cf. in 44.8 g C m−2 semi- and 68.3 g C m−2 in improved grasslands) and semi-improved grassland at Abergwyngregyn (76.0 g C m−2 cf. 41.7 g C m−2 in un- and 58.3 g C m−2 in improved grasslands). Cmic showed little temporal variation (v=3.7%). Soil microbial activity, measured as basal respiration was also mainly affected by grassland type and site (n=32%). In contrast to Cmic, respiration was significantly (P<0.001) high in the improved grassland at Sourhope (263.4 l h−1m−2 cf. 79.6 l h−1m−2 in semi- and 203.9 l h−1m−2 unimproved grasslands) and Abergwyngregyn (198.8 l h−1m−2 cf. 173.7 l h−1m−2 in semi- and 88.2 l h−1m−2 unimproved grasslands). Microbial activity, measured as potential carbon utilisation, agreed with the respiration measurements and was significantly (P<0.001) high in the improved grassland at all three sites (A590 0.14 cf. 0.09 in semi- and 0.07 in unimproved grassland). However, date of sampling also had a significant (P<0.001) impact on C utilisation potential (v=24.7%) with samples from April 1997 having highest activity at all three sites. Variation in microbial community structure was due, predominantly, to grassland type (average v=23.6% for bacterial and fungal numbers and PLFA) and date of sampling (average v=39.7% for bacterial and fungal numbers and PLFA). Numbers of culturable bacteria and bacterial PLFA were significantly (P<0.001) high in the improved grassland at all three sites. Fungal populations were significantly (P<0.01) high in the unimproved grassland at Sourhope and Abergwyngregyn. The results demonstrate a shift in soil microbial community structure from one favouring fungi to one favouring bacteria as grassland improvement increased. Numbers of bacteria and fungi were also significantly (P<0.001) higher in August than any other sampling date. Canonical variate analysis (CVA) of the carbon utilisation data significantly (P<0.05) differentiated microbial communities from the three grassland types, mainly due to greater utilisation of sugars and citric acid in the improved grasslands compared to greater utilisation of carboxylic acids, phenolics and neutral amino acids in the unimproved grasslands, possibly reflecting substrate availability in these grasslands. Differences in Cmic, activity and community structure between grassland types were robust over time. In addition, broad scale measures of microbial growth and activity (Cmic and respiration) showed little temporal variation compared to measures of soil microbial community structure, which varied quantitatively with respect to environmental variables (temperature, moisture) and plant productivity, hence substrate supply.