The role of AMP in photophosphorylation was studied using rapid mixing acid quench techniques. Fragmented spinach chloroplast membranes or subchloroplast particles were illuminated and rapidly mixed with [32P]orthophosphate and AMP at pH 7 for 10 ms to 60 s after which time perchloric acid was added to quench the reaction. ATP was found to be the primary and predominant nucleotide labeled. It was found that after illumination, an adenylate kinase-like activity carried out an AMP-dependent conversion of labeled ATP to labeled ADP which was inhibited by the presence of ADP. This reaction was characterized as being similar to chloroplast adenylate kinase in Mg2+ dependency and in sensitivity to phlorizin and tentoxin and distinct from chloroplast coupling factor 1. The small amounts of adenylate kinase activity present in fragmented well washed chloroplast membranes were found to be sufficient to carry out this rapid reaction. These results necessitated a reinterpretation of the earlier findings of Tiefert and Moudrianakis (Tiefert, M.A., and Moudrianakis, E.N. (1979) J. Biol. Chem. 254, 9500-9508) and no longer support the role of ADP as a phosphorylated intermediate in ATP synthesis.