The effect of pyrophosphate (PP(i)) on labeled nucleotide incorporation into noncatalytic sites of chloroplast ATP synthase was studied. In illuminated thylakoid membranes, PP(i) competed with nucleotides for binding to noncatalytic sites. In the dark, PP(i) was capable of tight binding to noncatalytic sites previously vacated by endogenous nucleotides, thereby preventing their subsequent interaction with ADP and ATP. The effect of PP(i) on ATP hydrolysis kinetics was also elucidated. In the dark at micromolar ATP concentrations, PP(i) inhibited ATPase activity of ATP synthase. Addition of PP(i) to the reaction mixture at the step of preliminary illumination inhibited high initial activity of the enzyme, but stimulated its activity during prolonged incubation. These results indicate that the stimulating effect of PP(i) light preincubation with thylakoid membranes on ATPase activity is caused by its binding to ATP synthase noncatalytic sites. The inhibition of ATP synthase results from competition between PP(i) and ATP for binding to catalytic sites.