Wild-type and mutant plants of barley (Hordeum vulgare L. cv. Maris Mink) lacking activities of chloroplastic glutamine synthetase (GS) and of ferredox-in-dependent glutamate synthase (Fd-GOGAT) were crossed to generate heterozygous plants. Crosses of the F2 generation containing GS activities between 47 and 97 of the wild-type and Fd-GOGAT activities down to 63 of the wild-type have been selected to study the control of both enzymes on photorespiratory carbon and nitrogen metabolism. There were no major pleiotropic effects. Decreased GS had a small impact on leaf protein and the total activity of ribulose-1,5-bisphosphate carboxylase-oxygenase (Rubisco). The activation state of Rubisco was unaffected in air, but a decrease in GS influenced the activation state of Rubisco in low CO2. In illuminated leaves, the amino-acid content decreased with decreasing GS, while the content of ammonium rose, showing that even small reductions in GS limit ammonium re-assimilation and may bring about a loss of nitrogen from the plants, and hence a reduction in protein and Rubisco. Leaf amino-acid contents were restored, and ammonium and nitrate contents decreased, by leaving plants in the dark for 24 h. The ratios of serine to glycine decreased with a decrease in GS when plants were kept at moderate photon flux densities in air, suggesting a possible feedback on glycine decarboxylation. This effect was absent in high light and low CO2. Under these conditions ammonium contents exhibited an optimum and amino-acid contents a minimum at a GS activity of 65 of the wild-type, suggesting an inhibition of ammonium release in mutants with less than 65 GS. The leaf contents of glutamate, glutamine, aspartate, asparagine, and alanine largely followed changes in the total amino-acid contents determined under different environmental conditions. Decreased Fd-GOGAT resulted in a decrease in leaf protein, chlorophyll, Rubisco and nitrate contents. Chlorophyll a/b ratios and specific leaf fresh weight were lower than in the wild-type. Leaf ammonium contents were similar to the wild-type and total leaf amino-acid contents were only affected in low CO2 at high photon flux densities, but mutants with decreased Fd-GOGAT accumulated glutamine and contained less glutamate.