Adenosine diphosphate (ADP) plays a critical role in platelet activation both by exogenous stimulation and the release of endogenous intracellular stores. As the platelet ADP receptor is not well defined, we have chosen to identify and characterize several cell lines that possess functional receptors for this nucleotide. Rat promegakaryoblasts (RPM), human erythroleukemia cells (HEL), U937, and K562 leukemia cells responded to ADP, as measured by a rapid increase in intracellular calcium. In the case of RPM cells, ADP was the only naturally occurring platelet agonist capable of eliciting this response. Binding studies with [3H]ADP and fixed cells showed 3.99 +/- 1.77 x 10(5) binding sites/cell for RPM cells (apparent dissociation constant [kd] = 7.75 +/- 2.3 x 10(-8) mol/L), 8.19 +/- 3.25 x 10(5) sites/cell for HEL cells (kd = 2.15 +/- 0.84 x 10(-7) mol/L, 1.15 +/- 0.23 x 10(6) sites/cell for U937 cells (kd = 2.20 +/- 0.53 x 10(-7) mol/L) and 5.39 +/- 2.80 x 10(5) sites/cell for K562 cells (kd = 1.37 +/- 0.39 x 10(-7) mol/L), Inhibition studies with unlabeled nucleotides and analogues showed that binding was approximately 85% specific and the inhibitory pattern was similar to that seen with mature platelets. The purine base adenosine resulted in little or no inhibition. These studies indicate that both human and rat hematopoietic cell lines possess intact ADP receptors and may be useful tools in future studies of the structure and function of this important platelet-activation system.