Primary cultures of human umbilical vein endothelial cells (HEC) developed extensive cytopathic changes and necrosis after high multiplicity infection with wild-type SV40 virus. Using the calcium co-precipitation technique, stable transformation was obtained with purified preparations of intact circular SV40 DNA and restriction endonuclease-derived linear DNA fragments containing the entire early gene region. Smooth muscle cells, isolated from the same blood vessels, showed neither cytopathic effects nor transformation after similar treatment with SV40 virus or DNA. The HEC cultures transformed by SV40 (SVHEC) expressed SV40-specific T (tumor) and Tr (transplantation) antigens, but not V (viral capsid) antigen. No evidence of infectious virus production was found upon co-cultivation with the CV-1 line of monkey kidney cells. Transformation resulted in markedly increased growth potential, loss of anchorage dependence and topoinhibition of growth, and a reduced serum requirement. Prolonged subcultivation was accompanied by chromosomal abnormalities and eventual "crisis". Transformed cells did not exhibit endothelial-specific organelles (Weibel-Palade bodies) or factor VIII antigen, but angiotensin-converting enzyme occasionally was detectable in SVHEC cultures. SV40-transformed human vascular endothelium, a nonfibroblast diploid cell type, may be useful in studies of oncogenesis and control of the differentiated state.