Smooth muscle cells (SMCs) cultured from the neointima of injured rat carotid arteries have a different shape and organization in vitro than SMCs from the uninjured media. The morphology of neointimal SMCs from adult rats strongly resembles that of a subset of medial SMCs from 12-day-old rat pups. In the present study, we show that adult carotid neointimal SMCs in vitro express the platelet-derived growth factor (PDGF)-B gene but have little or no PDGF a-receptor mRNA. In contrast, medial SMCs from contralateral uninjured carotids, grown and passaged under identical conditions, contain abundant PDGF α-receptor mRNA but little or no PDGF-B mRNA. Transcript levels for PDGF-A or PDGF α-receptor were not different in neointimal versus medial SMC cultures. The PDGF mRNA phenotype of adult neointimal SMCs strongly resembles that of an aortic medial SMC subset from newborn rat pups. Although intriguing, the differences in SMC phenotypes we observed in cell culture may depend on unique conditions in vitro and do not necessarily mean that analogous SMC diversity also exists in vivo. To address this question, we constructed and screened a SMC cDNA library for additional molecular markers of the common “pup-intimal” SMC phenotype. Two cDNA clones were identified whose cognate mRNA levels were developmentally regulated in rat aorta in vivo and were present at high levels in the adult carotid neointima formed 2 weeks after balloon catheter injury. Importantly, elevated levels of these two cognate mRNAs in carotid neointima compared with underlying media were maintained in cultures of neointimal versus medial SMCs in vitro. DNA sequence analysis indicated that the cDNA clones encoded rat tropoelastin and SS1procollagen (type I). These results provide further evidence that neointima formation in the adult rat carotid artery depends on reexpression of an SMC phenotype or subpopulation with special properties characteristic of earlier stages of artery wall development. Our studies to date indicate that two of these special properties are paracrine growth factor production and extracellular matrix synthesis.