Synthetic activity of collagen types was examined in cultured arterial smooth muscle cells during modulation from synthetic to contractile phenotype by treatment with dimethyl sulfoxide (DMSO). Smooth muscle cells of rabbit thoracic aorta cultured with a 1% supplement of DMSO for 8 days (DMSO group) predominantly exhibited cellular features of the contractile type with abundant microfilaments and a distinct basement membrane. Cultured cells in the DMSO group or in controls during stationary or subconfluent phase were labeled with [3H]proline for 24 h, and the samples including the cell layer and medium were analyzed. The incorporation of proline into bacterial collagenase digestible fractions was increased slightly in the DMSO group. Type analysis of the collagenous protein by SDS PAGE and subsequent fluorography disclosed a markedly increased ratio of type IV/I collagen and a slightly increased type V/l collagen ratio, as compared with those of controls. A decrease of type III collagen production in DMSO treated cells probably due to their lower cell density was also recognized. From these biochemical and morphological observations, it is suggested that increased synthesis of minor collagen types, particularly type IV collagen, is closely associated with smooth muscle phenotypic expression following DMSO treatment. Similar cellular events may occur in smooth muscle cells migrating into the intima during the process of arteriosclerosisin vivo.