Two monoclonal antimyosin antibodies, Western blotting experiments, and immunofluorescence procedures were used to investigate myosin isoform expression in normal and atherosclerotic aortas of adult rabbits. The SM-E7 antibody reacted with the two myosin heavy chain (MHC) isoforms of smooth muscle (SM) type (SM-MHC-1 and SM-MHC-2) expressed in the adult rabbit aorta. The NM-G2 antibody recognized an epitope shared by the nonmuscle (NM) myosin heavy chains (NM-MHC) present in fibroblasts, macrophages, lymphocytes, and platelets. Two smooth muscle cell (SMC) populations were identified in the medial layer of normal adult aorta, namely cells that contained SM myosin exclusively and cells that showed the coexistence of SM and NM myosin isoforms. The size of the cell population with double myosin isoform content increased markedly during experimental atherogenesis and represented by far the predominant SMC phenotype in the atherosclerotic plaque. Western blotting analysis performed on crude extracts from the atherosclerotic plaque showed the presence of SM-MHC-1 and NM-MHC isoforms in this tissue. Co-expression of SM and NM myosin at the molecular and the cellular level were found in aortic tissue during the early stages of development. These results indicate that in experimental atherosclerosis, the accumulation in the plaque of SMC with an "immature" pattern of myosin isoform expression is accompanied by similar modifications in the differentiation pattern of SMC of the underlying media.