Binding of pyrophosphate (PP(i)) to the three catalytic ('C') and three noncatalytic ('NC') nucleotide sites of Escherichia coli F1-ATPase was determined by fluorescence spectroscopy using mutant enzymes with tryptophan inserted specifically in either C sites (βY331W) or NC sites (αR365W). Fluorescence of the tryptophan is quenched on binding of nucleotide; PP(i) binding parameters were determined by competition with ATP or adenyl-5'-yl imidodiphosphate. It was found that MgPP(i) binds to each NC site with K(d) = 20 μM. In contrast, even at millimolar concentration, neither MgPP(i) nor free PP(i) showed significant binding to C sites. We confirmed that free PPi displaces nucleotide from C sites, but this was shown to be due to complexation of Mg2+ ions rather than to occupancy of the sites. MgPP(i) bound at NC sites was found not to affect ATP hydrolysis rates. From the data we propose a two-phase model for nucleotide binding at NC sites. In phase one, NC sites recognize the pyrophosphate 'end' of the nucleotide, which binds initially with K(d) similar to MgPP(i); in phase two, a slow conformational change occurs which tightly sequesters adenine nucleotide. Phase two does not occur with guanine nucleotide. This model explains the preference of NC sites for adenine nucleotides. P(i) (5 mM) did not bind to either C or NC sites.