Erythropoietin (EPO) induces endothelin expression in endothelial cells (EC) and has angiogenic effects. We investigated the intracellular signal transduction of EPO in EC and tested the hypothesis that the proliferative effects of EPO may be mediated by cytosolic calcium, changes in intracellular pH, or tyrosine phosphorylation. Cytosolic calcium and pH were measured with fura-2 and BCECF. Protein phosphorylation was assessed with 32P-labeled EC and two-dimensional (2D) gel chromatography. Tyrosine phosphorylation was measured using specific antityrosine antibodies and confocal microscopy. Proliferation was measured by thymidine incorporation and cell count. No effects of EPO on cytosolic calcium and pH were observed. In contrast, erythropoietin increased phosphorylation of 94, 70, 42, 40, 29 and 25 kDa proteins at five minutes and 60 minutes. Most of the early proteins were tyrosine phosphorylated. Confocal microscopy showed cytosolic as well as membrane-bound tyrosine phosphorylation in resting cells and an EPO-induced translocation of immunoreactivity to the nucleus. Immunostaining for the transcription factor STAT-5 showed that EPO induced a nuclear translocation of STAT-5. EPO 0.5, 2, and 4 U/ml increased proliferation, an effect that was prevented by incubation with the tyrosine kinase inhibitor genistein. We conclude that EPO induces proliferation in EC initially via tyrosine phosphorylation of six distinct proteins, and that the phosphorylation and nuclear translocation of the transcription factor STAT-5 is important for the effects of EPO on EC.