Chloramphenicol (CAP) was found to be a potent inhibitor of endogenous CO2 fixation of mesophyll chloroplasts having the C3 (Calvin-Benson) pathway of photosynthesis. Both D-threo and L-threo CAP were equally inhibitory, with 50% inhibition occurring at roughly 1.2 μg/ml. Light-dependent incorporation of [3H] leucine into protein was inhibited only by the D-threo isomer with 50% inhibition at 2.5 μg/ml and complete inhibition at 10 μg/ml. Carbon dioxide fixation in the presence of 2.5 mM dihydroxyacetone phosphate, which does not require NADPH or the enzymes involved in the reduction of 3-phosphoglycerate, was not inhibited by CAP. In in vitro enzyme assays, CAP was not inhibitory to phosphogylceromutase or triose phosphate dehydrogenase. CAP was found to inhibit oxygen evolution (H2O → NADP) and photophosphorylation by chloroplast membranes with the degree of inhibition dependent upon the concentration of NADP. CAP in the absence of NADP did not induce oxygen evolution or ATP synthesis. The site of inhibition by CAP is suggested to be after ferredoxin, since CAP had no effect on the Mehler reaction. It is proposed that CAP inhibits CO2 fixation by blocking the reduction of NADP.