Treatment of 3T3-L1 adipocytes with insulin (IC50 approximately 200 pM insulin) or insulin-like growth factor-1 (IC50 approximately 200 pM IGF-1) stimulates dephosphorylation of CCAAT/enhancer binding protein alpha (C/EBPalpha), a transcription factor involved in preadipocyte differentiation. As assessed by immunoblot analysis of one- and two-dimensional PAGE, insulin appears to dephosphorylate one site within p30C/EBPalpha and an additional site within p42C/EBPalpha. Consistent with insulin causing dephosphorylation of C/EBPalpha through activation of phosphatidylinositol 3-kinase, addition of phosphatidylinositol 3-kinase inhibitors (e.g. wortmannin) blocks insulin-stimulated dephosphorylation of C/EBPalpha. In the absence of insulin, wortmannin or LY294002 enhance C/EBPalpha phosphorylation. Similarly, blocking the activity of FKBP-rapamycin-associated protein with rapamycin increases phosphorylation of C/EBPalpha in the absence of insulin. Dephosphorylation of C/EBPalpha by insulin is partially blocked by rapamycin, consistent with a model in which activation of FKBP-rapamycin-associated protein by phosphatidylinositol 3-kinase results in dephosphorylation of C/EBPalpha. The dephosphorylation of C/EBPalpha by insulin, in conjunction with the insulin-dependent decline in C/EBPalpha mRNA and protein, has been hypothesized to play a role in repression of GLUT4 transcription by insulin. Consistent with this hypothesis, the decline of GLUT4 mRNA following exposure of adipocytes to insulin correlates with dephosphorylation of C/EBPalpha. However, the repression of C/EBPalpha mRNA and protein levels by insulin is blocked with an inhibitor of the mitogen-activated protein kinase pathway without blocking the repression of GLUT4 mRNA, thus dissociating the regulation of C/EBPalpha and GLUT4 mRNAs by insulin. A decline in C/EBPalpha mRNA and protein may not be required to suppress GLUT4 transcription because insulin also induces expression of the dominant-negative form of C/EBPbeta (liver inhibitory protein), which blocks transactivation by C/EBP transcription factors.