The expression of Insulin-Like Growth Factor-Binding protein-1 (IGFBP-1) is induced in rat liver by dexamethasone and glucagon (Dex/Gluc), and completely inhibited by 100 nM insulin. Various studies have implicated PI3 kinase, protein kinase B (Akt), phosphorylation of the transcription factors Foxo1/Foxo3, and the mammalian target of rapamycin (mTOR) in insulin's effect. In this study we examined insulin regulation of IGFBP-1 in both subconfluent and confluent hepatocytes. In subconfluent hepatocytes insulin inhibition of IGFBP-1 mRNA levels was blocked by inhibiting PI3 kinase activation and there was a corresponding inhibition of Foxo1/Foxo3 phosphorylation. In these same cells inhibition of the insulin effect by rapamycin occurred in the presence of insulin-induced Foxo1/Foxo3 phosphorylation. In confluent hepatocytes insulin could not activate the PI3 kinase - Akt - Foxo1/Foxo3 pathway but still inhibited IGFBP-1 gene expression in an mTOR dependent manner. In subconfluent hepatocytes the Ser/Thr phosphatase inhibitor Okadaic acid (OA) (100 nM) partially inhibited IGFBP-1 gene expression by 40% while not producing phosphorylation of either Akt or Foxo proteins. In contrast 1 nM insulin inhibited IGFBP-1 mRNA level by 40% and correspondingly activated Akt and Foxo1/Foxo3 phosphorylation to a level comparable to that observed with 100 nM insulin. These results suggest a potential role for a Ser/Thr phosphatase(s) in the regulation of IGFBP-1 gene transcription, which is not downstream of mTOR and is independent of Akt. In conclusion we have found that in rat liver insulin inhibition of IGFBP-1 mRNA levels can occur in the absence of the phosphorylation of Foxo1/Foxo3 whereas activation of the mTOR pathway is both necessary and sufficient.