The purification and characterization of uridine and thymidine, two previously isolated vascular endothelium effectors, are described. These two effectors were purified from all dialyzable fractions of fetal bovine serum (FBS), vitreous, and retina. The present study demonstrates the stimulatory characteristic of uridine and thymidine on the proliferation of aortic endothelial cells. The stimulation appears to link to their utilization by the cells in a dialyzed FBS-dependent manner. In the presence of optimal concentrations of uridine (10 microM), thymidine (0.8 microM), and dialyzed FBS (3 mg ml-1), a markedly enhanced vascular endothelial cell proliferation is attainable; uridine and thymidine alone had no significant stimulatory effect. The presence of both nucleosides, however, increased the concentration of dialyzed FBS required for half-maximum proliferation activity of fetal bovine aortic endothelial cells and also increased maximum activity. Studies with fetal bovine aortic endothelial cells showed that the activity of uridine kinase in the cells was activated by dialyzed FBS, but not by uridine and thymidine. Uridine and thymidine exerted no marked stimulatory effect on Balb/c 3T3, Swiss 3T3, and SV-T2 as noted with endothelial cells. The significance of the present findings to the proliferation of vascular endothelial cells in the retina is discussed.