The possible involvement of the protein kinase C (PKC) pathway in transcriptional regulation of the human insulin-like growth factor-I (IGF-I) gene has been suggested. In this study, we sought to determine whether a PKC-dependent pathway is implicated in the transcriptional control, and if it is, how this occurs. Treatment with 12-O-tetradecanoylphorbol 13-acetate (TPA) caused an increase in the activity of the human IGF-I gene major promoter in HepG2 cells. A CCAAT/enhancer-binding protein (C/EBP) binding site located at +22 to +30 was bound by C/EBP beta in a TPA-dependent manner and was solely responsible for the TPA responsiveness. This increase in C/EBP beta activity occurs through transcriptional and posttranslational regulation, and the latter is mediated by activation of p90 ribosomal S6 kinase (RSK): co-expression of dominant negative RSK abolished the TPA-responsive and C/EBP beta-dependent transactivation. Also, TPA-responsive activation of GAL4-C/EBP beta chimera required the Ser residue known as the RSK target. In SK-N-MC cells, which display constitutive, high expression of IGF-I on use of the major promoter, a large amount of C/EBP beta binding was observed with the C/EBP site in the basal state. Treatment with PKC inhibitors substantially reduced the promoter activity and mRNA amounts of IGF-I, with the binding of C/EBP beta to the C/EBP site also being reduced. When the C/EBP site was disrupted, the basal promoter activity was reduced, but the reduction by the PKC inhibitor was no longer observed. These observations suggest that the increase of C/EBP beta binding to the C/EBP site, which is in part mediated via activation of RSK, can primarily explain the TPA responsiveness of the IGF-I gene promoter. The intrinsic PKC activity in SK-N-MC cells should play a major role in the constitutive, high expression of IGF-I and may therefore contribute in part to the maintenance of the tumor phenotype of the cells.