Several observations suggest the expansion of a distinct medial smooth muscle cell (SMC) subset in atherosclerosis and restenosis. We characterized the phenotypic features of SMC subsets in cultures derived from human carotid endarterectomy specimens. Specimens comprised an undiseased portion (thin intimal thickening with the underlying media) and a diseased portion (atherosclerotic plaque with the underlying media). From plaque tissues of the diseased portion, only macrophage-derived foam cells were retrieved. From medial tissues, two SMC phenotypes were isolated: large SMCs (flat with a monolayered growth pattern, from the undiseased portion) and small SMCs (fusiform and growing in multilayers, from the undiseased and diseased portions after co-culture with macrophage-derived foam cells). Small SMCs displayed higher proliferative and migratory activities and were less differentiated than large SMCs. Proteomic analysis showed that calmodulin was predominant in small SMCs. Co-culture of large SMCs with macrophage-derived foam cells induced a transition to the small phenotype with increased calmodulin expression. The calmodulin inhibitor W-7 decreased the proliferation of small SMCs and prevented the large to small phenotypic transition. In vivo, calmodulin was markedly expressed in SMCs of atherosclerotic plaques and was barely detectable in the media. Macrophage-derived foam cells promote selective migration from the media of atheroma-prone SMCs characterized by calmodulin overexpression. Further studies of small SMCs could be instrumental in understanding atherosclerosis pathogenesis and in planning therapeutic strategies.