Intact chloroplasts of wheat (Triticum aestivum) were isolated from mesophyll protoplasts. With decreasing concentrations of bicarbonate from 10 to 0.3 millimolar (pH 8.0), the optimal concentration of orthophosphate (Pi) for photosynthetic O(2) evolution decreased from a value of 0.1 to 0.2 millimolar to 0 to 0.025 millimolar. The extremely low Pi optimum for photosynthesis at the low bicarbonate levels of 0.3 millimolar was increased by lowering the O(2) concentration from 253 (21% gas phase) to 72 micromolar (6% gas phase). The relative amount of glycolate and dihydroxyacetone phosphate (DHAP) synthesized under high and low levels of bicarbonate and varying levels of Pi was determined. At low levels of bicarbonate, glycolate was the main product, whereas at high bicarbonate levels, DHAP was the main product. Most of the DHAP and glycolate was found in the extrachloroplastic fraction.The rate of photosynthesis at low levels of bicarbonate (0.3 millimolar) was as high as 75 to 95% of that at high levels of bicarbonate (10 millimolar) at the respective optimal levels of Pi. At low bicarbonate levels, and without Pi, there was little lag in photosynthetic O(2) evolution upon illumination in comparison to that of high bicarbonate levels and optimal levels of Pi. It is proposed that conditions which favor glycolate synthesis allow photosynthesis to continue without a depletion of internal Pi, whereas consumption of Pi may occur in the chloroplast during the net synthesis of organic phosphates under high levels of bicarbonate and without addition of Pi. At low bicarbonate levels, the extreme susceptibility of photosynthesis to inhibition by Pi may be due to excessive export of carbon from the chloroplast in the form of both glycolate and triose phosphate.