Different smooth muscle cell (SMC) functions may require different cell phenotypes. Because the main pulmonary artery performs diverse functions, we hypothesized that it would contain heterogeneous SMC populations. If the hypothesis were confirmed, we wished to determine the developmental origin of the different populations. Using specific antibodies, we analyzed the expression of smooth muscle (SM) contractile and cytoskeletal proteins (alpha-SM-actin, SM myosin, calponin, desmin, and meta-vinculin) in the main pulmonary artery of fetal (60 to 270 days of gestation), neonatal, and adult animals. We demonstrated the existence of a complex, site-specific heterogeneity in the structure and cellular composition of the pulmonary arterial wall. We found that at least four cell/SMC phenotypes, based on immunobiochemical characteristics, cell morphology, and elastic lamellae arrangement pattern, were simultaneously expressed within the mature arterial media. Further, we were able to assess phenotypic alterations in each of the four identified cell populations during development. We found that each cell population within the arterial media expressed alpha-SM-actin at least at certain stages of development, thus demonstrating its smooth muscle identity. However, each cell population progressed along different developmental pathways, suggesting the existence of multiple and distinct cell lineages. A novel anti-metavinculin antibody described in this study reliably distinguished one SMC population from the others during all the developmental stages analyzed. We conclude that the pulmonary arterial media is indeed composed of multiple phenotypically distinct cell/SMC populations with unique lineages. We speculate that these distinct cell populations may serve different functions within the arterial media and may also respond in unique ways to pathophysiological stimuli.