The role of photorespiration in the foliar assimilation of nitrate (NO3–) and carbon dioxide (CO2) was investigated by measuring net CO2 assimilation, net oxygen (O2) evolution, and chlorophyll fluorescence in tomato leaves (Lycopersicon esculentum). The plants were grown under ambient CO2 with ammonium nitrate (NH4NO3) as the nitrogen source, and then exposed to a CO2 concentration of either 360 or 700 µmol mol−1, an O2 concentration of 21 or 2%, and either NO3– or NH4+ as the sole nitrogen source. The elevated CO2 concentration stimulated net CO2 assimilation under 21% O2 for both nitrogen treatments, but not under 2% O2. Under ambient CO2 and O2 conditions (i.e. 360 µmol mol−1 CO2, 21% O2), plants that received NO3– had 11–13% higher rates of net O2 evolution and electron transport rate (estimated from chlorophyll fluorescence) than plants that received NH4+. Differences in net O2 evolution and electron transport rate due to the nitrogen source were not observed at the elevated CO2 concentration for the 21% O2 treatment or at either CO2 level for the 2% O2 treatment. The assimilatory quotient (AQ) from gas exchange, the ratio of net CO2 assimilation to net O2 evolution, indicated more NO3– assimilation under ambient CO2 and O2 conditions than under the other treatments. When the AQ was derived from gross O2 evolution rates estimated from chlorophyll fluorescence, no differences could be detected between the nitrogen treatments. The results suggest that short-term exposure to elevated atmospheric CO2 decreases NO3– assimilation in tomato, and that photorespiration may help to support NO3– assimilation.