Endothelial cell migration and proliferation are the key steps in the angiogenic process, and both are stimulated by recombinant human erythropoietin (rHuEPO). In addition rHuEPO can increase endothelin-1 (ET-1) release by the endothelial cell. We designed the present study to address the question of whether rHuEPO stimulates angiogenesis. An in vitro quantitative assay for angiogenesis was used. This consisted of rat aortic rings embedded in a reconstituted basement membrane matrix and incubated with and without rHuEPO for eight days. We found that rHuEPO increased vessel outgrowth after four days of culture and this was continued for the next four days (rHuEPO vs. control: day 4, 12 ± 2 vs. 4 ± 1, P < 0.002 and day 8, 124 ± 18 vs. 56 ± 12 P < 0.006). Supernatant endothelin-1 (ET-1) levels, at 24 hours, were significantly higher than controls in the rings incubated with rHuEPO (107 ± 13 vs. 43 ± 10 pg/ml, P < 0.003). To investigate the role of ET-1 in rHuEPO-induced angiogenesis, rings were exposed to ET-1 alone (10-8 M). We observed an increase in microvessel formation compared to control (day 4, 4 ± 2 vs. 2 ± 1, P < 0.006, and day 8, 67 ± 12 vs. 51 ± 10, P < 0.03). In addition, aortic rings were co-cultured with rHuEPO and anti-ET-1 IgG antibody. Stimulation of angiogenesis by rHuEPO was blunted by the ET-1 antibody. We conclude that rHuEPO stimulates angiogenesis in vitro, and this effect is due at least in part to the enhanced autocrine release of ET-1 by rHuEPO.