Vascular endothelial growth factor (VEGF) occurs in at least five different isoforms because of alternative splicing of the gene. To investigate the roles of different VEGF isoforms in tumor blood vessel formation and tumorigenicity, the three major isoforms (VEGF(121), VEGF(165), and VEGF(189)) were overexpressed in an early-stage human melanoma cell line (WM1341B), which is VEGF-negative and nontumorigenic in immunodeficient mice. Although overexpression of VEGF(121) and VEGF(165) resulted in aggressive tumor growth, WM1341B cells transfected with VEGF(189) remained nontumorigenic and dormant on injection. Although tumor growth rate depended on the level and not the isoform of VEGF expressed, striking isoform-specific differences in vascular patterning were associated with VEGF(121)- versus VEGF(165)-dependent tumorigenic conversion of human melanoma. Thus, tumors overexpressing VEGF(165) generated dense, highly heterogeneous vessel networks that were distinctly different from those of tumors expressing VEGF(121) (poorly vascularized and necrotic). Paradoxically, although VEGF(165) expression appears to result in the most effective tumor perfusion, it is the expression of VEGF(121) that is observed during human malignant melanoma progression. Indeed, unbiased selection of spontaneously tumorigenic variants of WM1341B (by coinjection with Matrigel) led to predominant expression of the VEGF(121) isoform. The vascular patterning in these tumors (1341-P3N1, 1341-P3N2) resembled that of the VEGF(121)-transfected WM1341B tumors. These results suggest that, for reasons that remain to be elucidated, a "minimal" program of tumor vascularization may be favored during melanoma progression.