In rat liver derived HTC cells transfected with and expressing human insulin receptors, there are multiple p60-79 proteins that are tyrosine phosphorylated following insulin treatment of cells. Employing antibodies to insulin receptor substrate 3 (alpha-IRS-3), we found that IRS-3 is a major p60 phosphoprotein that is tyrosine phosphorylated following insulin treatment of cells and interacts with phosphatidylinositol-3-kinase (PI3K). Majority of IRS-3 when phosphorylated appears to interact with PI3K. Tyrosine phosphorylation of IRS-3 is robust at 2 min and steadily increases up to 30-90 min of insulin treatment. Following insulin treatment of cells, some high molecular weight phosphoproteins are coimmunoprecipitated with alpha-IRS-3. In summary, IRS-S is the major p60 protein that is tyrosine phosphorylated and interacts with PI3K in HTC rat liver derived cells following insulin treatment of cells. Unlike related IRS-1/2 that is transiently phosphorylated, IRS 3 shows robust and prolonged tyrosine phosphorylation upon insulin treatment of cells and may play a role in delayed and/or prolonged insulin actions.