Human arterial smooth muscle cells transfected with the plasmid pSV3-neo, which contains the SV40 virus early region and the neor gene, developed colonies of morphologically transformed cells. Five cell strains were initiated from these colonies and could be subcultivated for up to 9 months before entering a stage of crisis that ended their life span. Deoxyribonucleic acid (DNA) molecules containing viral sequences were found free and integrated in the transformed cells. The intranuclear SV40 large T antigen and the p53 cellular protein were expressed in the transformed cells. Most of the transformed cells were spindle shaped but some were large and multinucleated. The modal chromosome numbers were in the triploid range, and aberrations, particularly dicentrics, were common. The transcripts for smooth muscle actins were significantly reduced and there were less alpha-actin filaments detected by immunofluorescence. Cytochemical staining disclosed a large accumulation of lipid droplets in the transformed cells incubated with rabbit hypercholesterolemic beta-very-low-density lipoprotein. Chemical analysis showed that cholesteryl esters were significantly elevated in these cells. Phenotypic changes induced in human vascular smooth muscle cells by SV40 early genes are similar to those found in smooth muscle cells from atherosclerotic lesions and may indicate common pathogenetic mechanisms.