In neutrophils and several other phagocytes, a pertussis and cholera toxin-sensitive guanine nucleotide-binding protein (G-protein) couples the receptors for formyl methionine-containing chemotactic peptides to stimulation of phospholipase C. We used membranes of myeloid-differentiated HL 60 cells to study the role of Na+ in regulating both the interaction of the formyl peptide receptor with the chemotactic agonist, N-formyl-methionyl-leucyl-phenylalanine (FMLP), and the receptor-mediated activation of the G-protein. Monovalent cations (Na+ greater than Li+ greater than K+ greater than choline+) markedly inhibited the binding of the radiolabeled oligopeptide [3H]FMLP by specifically reducing the number of receptors in the high-affinity state. Half-maximal and maximal inhibition of peptide binding were seen at cation concentrations of approximately 20 and 200 mM, respectively. Inhibition of peptide binding by Na+ was observed in the presence and absence of divalent cations and was strictly additive to inhibition by the poorly hydrolyzable GTP analogue, guanosine-5'-O-(3-thiotriphosphate), or to ADP ribosylation of G-proteins by pertussis toxin. The inhibitory effect of Na+ on peptide binding coincided with a marked reduction of the potency of FMLP to stimulate a high-affinity GTPase. In contrast, the degree of FMLP-stimulated GTPase activity was markedly enhanced in the presence of Na+. This was largely due to the fact that Na+ reduced the agonist-independent basal GTPase activity in the same way but less so than pertussis toxin treatment. The results show that monovalent cations, Na+ in particular, regulate the interaction of the formyl peptide receptor with both the chemotactic agonist and the G-protein by acting on a single site, possibly located on the receptor itself. The observation that basal GTPase activity is markedly reduced by both Na+ and pertussis toxin treatment also suggests (a) that G-proteins interact with and are activated by receptors even in the absence of agonists and (b) that Na+ uncouples unoccupied receptors from G-protein interaction and activation.