Insulin has been shown to be important for normal liver regeneration to occur. The mechanisms whereby insulin may exert its effects on hepatocyte growth, however, are still unknown. The rat and human insulin receptor substrate 1 (IRS-1) is a specific target molecule for the insulin receptor beta subunit kinase and will bind to signal transducing molecules containing Src homology 2 domains through its multiple tyrosyl phosphorylation (TP) sites. This investigation examined how IRS-1 may be involved in insulin action during hepatocyte growth induced by two-thirds partial hepatectomy. The TP of IRS-1 was strikingly enhanced prior to the major wave of hepatocyte DNA synthesis at 24 h; IRS-1 protein and mRNA expression increased in parallel to a lesser extent after partial hepatectomy. TP of insulin receptor beta subunit, which enhances its kinase activity toward IRS-1, was increased in association with TP of IRS-1. Finally, phosphatidylinositol 3-kinase, one of signal transducing molecules containing Src homology 2 domains, was associated with IRS-1 following TP in vivo. These observations suggest that IRS-1 protein may play an important role in transmitting the insulin signal to intracellular regulators involved in hepatocyte growth.