The effects of guanylates and inosinates (and adenylates) on phosphorylation, ferricyanide reduction, and light-induced H+ uptake in spinach chloroplasts were studied. GDP, GTP, IDP, and ITP (but not GMP and IMP) stimulated the light-induced H+ uptake and partially inhibited ferricyanide reduction. Phosphate, arsenate, and phlorizin increased the extent of inhibition by these nucleotides and decreased the values of their apparent dissociation constants for the inhibition process. In the presence of phosphate (or arsenate), restoration of ferricyanide reduction from the level inhibited by guanylates and inosinates was observed as phosphorylation (or arsenylation) proceeded. These results suggest that phosphorylation of GDP and IDP as well as ADP takes place after two steps of nucleotide binding to the chloroplast coupling factor 1. The apparent dissociation constants of GDP and IDP for these two binding steps were estimated to be about 34 and 38 muM for the first and 110 and 160 muM for the second step, respectively (at pH 8.3, 15 degrees C). Above pH9, the ratio (P/delta-e) of the extent of phosphorylation to the increment of electron transport from the basal level measured in the presence of [ATP + Pi] or [ADP + Pi+ phlorizin], became increasingly large. When the electron transport level inhibited by dicyclohexyl-carbodiimide was taken to be the basal activity, the P/delta-e ratio remained almost constant (approximately 1) from pH 7.0 up to 10.