The mechanisms whereby P2-purinergic receptors for extracellular ATP are coupled to the inositol phospholipid-signaling system were studied in the HL60 human promyelocytic leukemia cell line. Brief pretreatment of either undifferentiated or differentiated HL60 cells with various activators of protein kinase C Ca2+/phospholipid-dependent enzyme (e.g. phorbol myristate acetate) produced a 50-fold decrease in the potency of extracellular ATP to induce mobilization of intracellular Ca2+. The ATP-induced increase in rate of inositol trisphosphate (InsP3) accumulation in these 4-beta-phorbol 12-myristate-13-acetate-treated cells was characterized by a 40% decrease in the maximal rate of InsP3 accumulation. Incubation of the cells with NaF also induced mobilization of the same Ca2+ stores released in response to extracellular ATP; this provided indirect evidence that the transmembrane signaling actions of P2-purinergic receptors may be mediated by GTP-binding regulatory proteins. This latter possibility was further supported by the finding that treatment of either undifferentiated or differentiated HL60 cells with pertussis toxin produced a significant, but partial, inhibition of ATP-induced signaling actions. These included: 1) a 60-70% decrease in the maximum rate of InsP3 accumulation, and 2) a 1.5 log unit increase in the half-maximally effective [ATP] required for mobilization of intracellular Ca2+. In cells treated with both pertussis toxin and 4-beta-phorbol 12-myristate-13-acetate, there was an 80% decrease in maximal rate of ATP-induced InsP3 accumulation and near-complete inhibition of ATP-induced Ca2+ mobilization. Significantly, the residual, pertussis toxin-insensitive portion of ATP-induced signaling was observed in the same samples of differentiated HL60 cells wherein pertussis toxin treatment produced complete abolition of InsP3 accumulation and Ca2+ mobilization in response to occupation of chemotactic peptide receptors. These results indicate that the activation of inositol phospholipid breakdown by P2-purinergic receptors in HL60 cells may be mediated by both pertussis toxin-sensitive and toxin-insensitive mechanisms; this suggests that these myeloid progenitor cells may express two distinct types of GTP-binding proteins coupled to phospholipase C.