The rates of respiration in light and darkness, C i/C a and carbon isotope fractionation were investigated in glycine decarboxylase-deficient plants of barley, potato and Arabidopsis thaliana grown in climate chambers with controlled light intensity, temperature, humidity, irradiation and different CO2 concentrations (360, 700 and 1400 µl l–1) and compared to the wild-type plants. All photorespiration-impaired plants exhibited higher C i/C a and corresponding lower apparent water-use efficiencies, which were more expressed under high irradiance and elevated temperature. The mutants were depleted in 13C as compared to the wild-type plants, with a difference of up to 6 following growth in 360 µl l–1 CO2. We determined the carbon isotope content at different CO2 concentrations to calculate the contribution of both C i/C a and photorespiration for 13C/12C fractionation. The direct effect of photorespiration was in the range of 0.7–1.0, from which we calculated the value of fractionation at the site of glycine decarboxylation as being 10–13, which is in agreement with the previously reported carbon isotope discrimination exerted by the glycine decarboxylase. Respiratory rates, particularly in the light, were increased in the glycine decarboxylase mutants. The necessity of the maintenance of a high CO2 concentration near the site of carboxylation in chloroplasts in plants deficient in photorespiratory enzymes, requires an increased opening of the stomata with a corresponding decrease in water-use efficiency. It is concluded that photorespiration participates in the regulation of C i/C a and contributes to carbon isotope fractionation, both via effects on stomata and via discrimination of 13C in the glycine decarboxylase reaction.