In granulocytes, emptying of intracellular Ca2+ stores activates Ca2+ influx across the plasma membrane. To study the putative role of GTP-binding proteins in this process, we have introduced non-hydrolyzable guanosine phosphate analogues into the cytosol of non-permeabilized HL-60 granulocytes using an endocytosis-hypoosmotic shock procedure. At the cytosolic concentrations obtained (100-500 microM), neither guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) nor guanosine 5'-3-O-(thio)diphosphate (GDP beta S) affected basal [Ca2+]i. Ca2+ release in response to the receptor agonist fMet-Leu-Phe, the Ca(2+)-ATPase inhibitor thapsigargin, or the Ca2+ ionophore ionomycin was also unaffected by GTP gamma S or GDP beta S. In contrast, the activation of the Ca2+ influx pathway by fMet-Leu-Phe or by thapsigargin was blocked by GTP gamma S but not by GDP beta S. The GTP gamma S effect was mimicked by NaF. The GTP gamma S and NaF effects were independent of protein kinase C activation and actin polymerization. Our results demonstrate that a GTP-sensitive element is involved in the signaling between intracellular Ca2+ stores and plasma membrane Ca2+ channels. The identical effects of GTP gamma S and NaF suggest that the GTP-sensitive element is a heterotrimeric G-protein.