The purpose of this study was to identify and optimize the antiangiogenic activity of IFN-α against human bladder cancer cells growing in the bladder of nude mice. 253J B-V IFN^R cells (resistant to antiproliferative effects of IFN-α or IFN-β) were implanted into the bladder wall of nude mice. Three days later, the mice were treated with s.c. injections of IFN-α (70,000 units/week) at different dosing schedules (1, 2, 3, or 7 times/week). Daily therapy with IFN-α produced the most significant inhibition of tumor growth, tumor vascularization, and down-regulation of basic fibroblast growth factor and matrix metalloprotease-9 mRNA and protein expression. Changing dose and schedule of IFN-α administration had minimal effects on the expression of vascular endothelial growth factor or interleukin 8. The daily s.c. administrations of 5,000 or 10,000 units IFN-α-2a produced maximal inhibition of bFGF and MMP-9 expression (mRNA and protein), maximal reduction in tumor vessel density, and maximal reduction in serum levels of bFGF. Daily administration of higher doses of IFN-α failed to produce significant antiangiogenic effects. These data suggest that the antiangiogenic activity of IFN-α is dependent on frequent administration of optimal biological dose and not maximal tolerated dose.