Attached leaves of sunflower (Helianthus annuus L.) were exposed to (14)CO2 during steady-state photosynthesis for 2 to 30 min in 345 μl/l CO2 and 21% O2 at 29° C and a light intensity of 1300 μE m(-2)s(-1). Glycolic acid was extracted with water and diethyl ether, and was determined in the aqueous residue by high-pressure liquid column chromatography. The relative specific radioactivity of the glycolic acid synthesized during photosynthesis reached about 100% after 30 min of photosynthesis and was almost equal to that of the CO2 evolved during photorespiration, their ratio at all times being nearly one. These results provide strong in-vivo evidence that the glycolic acid is the substrate for CO2 evolved by sunflower leaves in light.