Aims: A laboratory assay for comparative characterization of various faecal matrices with respect to faecal indicator organism (FIO) release using, artificial rain water. Methods and Results: Fresh sheep and beef-cattle faeces, dairy cattle slurry and beef cattle farm yard manure (FYM) were collected from commercial units in south-west England and applied to 20 randomized 1 m2 plots established on permanent grassland. Representative samples from each faecal matrix (n = 5) were collected on four occasions over 16 days. One gram of each sample was transferred to a sterile vial to which 9 ml of standard local rain was carefully pipetted. The vial was then rotated through 360, 20 times in 60 s to ‘simulate’ a standardized interaction of the faecal material with rainfall, providing an assay of comparative release potential. Appropriate decimal dilutions were prepared from the eluent. Following agitation, with a sterile spatula, the remaining faecal material and eluent in the vials were vortex mixed for 60 s before decimal dilutions were prepared from the resulting mixture, providing a quantitative assessment of the total FIO in the sample from which percentage release could be determined. Bacterial concentrations were enumerated in duplicate by membrane filtration following standard methods for FIO. Significant differences in release kinetics of Escherichia coli and enterococci from each of the faecal matrices were determined. Conclusions: Differences in release from each faecal substrate and between FIO type (E. coli and intestinal enterococci) were observed in this laboratory study. The order of release of E. coli from the faecal matrices(greatest to least, expressed as a percentage of the total present) was dairy cattle slurry > beef cattle FYM > beef-cattle faeces > sheep faeces. For intestinal enterococci the order of percentage release was dairy cattle slurry > beef-cattle faeces > beef cattle FYM > sheep faeces. Significance and Impact of the Study: This laboratory-based method provides the first data on the relative release kinetics of FIO from different faecal matrices in rain water. This is fundamental information needed to parameterize laboratory-based microbial models and inform approaches to field and catchment risk assessment.