Clones were derived by dilute plating from cultured aortic smooth muscle cells of 12-day-old rats. Clones Pup I to III resemble uncloned smooth muscle cultures from aortas of rat pups and from adult rat neointimas. They have a cobblestone morphology and proliferate in plasma-derived serum. By Northern analysis they contain platelet-derived growth factor B chain (PDGF-B) mRNA and high levels of CYPIA1, elastin, and osteopontin mRNAs, and they lack platelet-derived growth factor alpha-receptor (PDGF-alpha R) mRNA. In contrast, Pup V resembles smooth muscle cultures derived from uninjured adult rat arteries. It has an elongated morphology and proliferates poorly in plasma-derived serum. This clone expresses PDGF-alpha R mRNA, low levels of elastin and osteopontin mRNAs, and lacks CYPIA1 and PDGF-B mRNAs. Pup IV and VI have most of the properties of Pup I to III. We conclude that uncloned pup cultures are heterogeneous, but that the growth properties and gene expression pattern described for the uncloned culture is characteristic of individual clones within the population.