Transfection of primary rat fetal brown adipocytes with constructs of SV40 large T antigen, alone and together with lys12-mutated H-ras gene, gave permanent cell lines showing an immortalized or transformed phenotype, respectively, all of them selected by the expression of the uncoupling protein (UCP), a tissue-specific marker. Primary brown adipocytes and immortalized cell lines respond to insulin-like growth factor I (IGF-I) by increasing their lipid content and the mRNA expression of both the adipogenic marker fatty acid synthase (FAS) and the thermogenic marker UCP. IGF-I-induced differentiation-related gene expression at 24 h in both primary and immortalized brown adipocytes was mediated by an increase in p21ras.GTP active protein content. Transformed cell lines overexpressing exogenous p21ras (mainly in its ras.GTP active form) constitutively showed a higher lipid content and a higher FAS and UCP mRNA expression compared to primary and immortalized cells. These transformed cells were IGF-I independent with respect to their studied differentiation-related parameters. Additionally, transient transfection of primary brown adipocytes with the transforming ras gene induced UCP and FAS mRNA expression as well as cotransactivated UCP-chloramphenicol acetyltransferase fusion gene. Moreover, IGF-I transactivation of UCP promoter was partially precluded by cotransfection with the dominant-negative ras gene. Our results strongly suggest that IGF-I/p21ras induces adipogenic- and thermogenic-related gene expression in brown adipocytes.