In order to study whether phosphate transfer reactions are involved in the binding of guanine nucleotide triphosphates to guanine-nucleotide-binding regulatory proteins, binding of the GTP analogues, guanosine 5'-[gamma-thio]triphosphate, GTP[S], and guanosine 5'-[beta, gamma-imino]triphosphate, p[NH]ppG, and the regulation of binding by the formyl-peptide-receptor agonist, fMet-Leu-Phe, were studied in membranes of differentiated HL-60 cells. For fMet-Leu-Phe-stimulated binding of either GTP analogue, a competing nucleotide was required. With GDP as the competing nucleotide, initial rates of fMet-Leu-Phe-stimulated binding of GTP[S] and p[NH]ppG were similar for up to approximately 30 s. Thereafter, receptor-stimulated binding of p[NH]ppG rapidly reached equilibrium, whereas the binding of GTP[S] proceeded further. At equipotent concentrations of p[NH]ppG and GTP[S], maximal fMet-Leu-Phe-stimulated binding of GTP[S] was approximately twofold higher than that of p[NH]ppG. Finally, for half-maximal receptor-stimulated binding of GTP[S], approximately fivefold higher concentrations of both Mg2+ and GDP were required than for p[NH]ppG binding. With p[NH]ppG as the competing nucleotide, the extent of receptor-stimulated binding of GTP[S] as well as its Mg2+ requirement and time course were similar to the receptor-stimulated p[NH]ppG binding observed in the presence of GDP. However, with GTP[S] as the competing nucleotide, fMet-Leu-Phe reduced the binding of p[NH]ppG, a reaction further enhanced when GDP was additionally present. Under similar conditions as used in the binding studies, GTP[S] thiophosphorylated a 35-kDa protein, which is most likely a guanine-nucleotide-binding regulatory protein beta subunit [Wieland, T., Nürnberg, B., Ulibarri, I., Kaldenberg-Stasch, S., Schultz, G. & Jakobs, K. H. (1993) J. Biol. Chem. 268, 18111-18118]. The thiophosphorylation state of this protein was regulated by guanine nucleotides, Mg2+ and, most importantly, by activated formyl-peptide receptors. The data thus provide evidence for an essential difference between GTP[S] and p[NH]ppG binding to guanine-nucleotide-binding regulatory proteins and suggest that, in addition to the nucleotide-exchange reaction, a (thio)phosphate-group-transfer process via guanine-nucleotide-binding regulatory protein beta subunits is involved in the receptor-stimulated binding of guanine nucleotide triphosphates to guanine-nucleotide-binding regulatory proteins.