In previous studies we have demonstrated that extracellular ATP (and UTP), acting through P2-purinergic receptors, can stimulate the inositol phospholipid signaling system in neutrophils and monocytes, as well as in neutrophil/monocyte progenitor cells. In this study we have examined the ability of extracellular nucleotides to modulate the phenotype of myelomonocytic progenitor cells. As model systems, we utilized the established HL-60 promyelocytic and U937 promonocytic human cell lines which were cultured in the continuous presence of nucleotides known to be potent agonists for P2-purinergic receptors. When cultured for 5 days with ATP gamma S (a phosphatase resistant analog of ATP) plus 10% fetal bovine serum, both HL-60 cells and U937 cells expressed several (but not all) phenotypic characteristics of differentiated phagocytes. In HL-60 cells these characteristics were (1) increased intracellular calcium mobilization in response to formylated chemotactic peptides, (2) a reduction in cell size with a decreased nuclear/cytoplasmic ratio, (3) a sharply reduced rate of proliferation, (4) a reduction in the percentage of cells expressing surface transferrin receptors, and (5) an increase in the percentage of cells expressing the type 1 complement receptor (CR1). In U937 cells these characteristics were (1) increased intracellular calcium mobilization in response to formylated chemotactic peptides and platelet activating factor, (2) a reduced rate of proliferation, (3) a reduction in the percentage of cells expressing surface transferrin receptors, and (4) increases in the percentage of cells expressing both type 1 (CR1) and type 3 (CR3) complement receptors. During the first 12-24 hr after exposure to ATP gamma S, HL-60 cells showed no obvious changes in morphology, viability, or the levels of beta-actin mRNA, but did show (1) a 4-fold increase in chemotactic peptide-induced Ca2+ mobilization, and (2) a greater than 90% decrease in c-myc mRNA levels. Significantly, when HL-60 cells were treated under serum-free conditions, the ability of ATP to enhance expression of functional FMLP receptors could be dissociated from the inhibitory effects of adenine nucleotides on cell proliferation observed in serum containing media. Moreover, treatment of serum-free HL-60 cultures with UTP, another P2-purinergic receptor agonist, also resulted in enhanced expression of functional FMLP receptors.