The insulin receptor, following insulin stimulation of cells, triggers formation of various signaling complexes. In rat HTC hepatoma cells overexpressing normal human insulin receptors (HTC-IR), p85 regulatory subunit of phosphatidylinositol-3-kinase (PI3K) forms signaling complexes containing the insulin receptor, insulin receptor substrate 1 (IRS-1), guanosine triphosphatase-activating protein (GAP) and 60-70 kDa phosphotyrosine proteins (p60-70). In the present study, we demonstrate that p60-70 interacts directly with the p85 subunit via src homology 2 domain of the latter. Employing antibodies specific to two p85 isoforms, p85alpha and p85beta, we demonstrate that HTC-IR cells express both p85 isoforms, and these isoforms induce the formation of similar signaling complexes in response to insulin. p60-70, present in both alpha-p85alpha and alpha-p85beta immunoprecipitates, is a GAP-associated protein, but is distinct from the p68 src-associated protein in mitosis (Sam68) by several criteria. These data suggest that 1) GAP-associated protein, but not Sam68, is a part of insulin signaling complexes; and 2) p85alpha and p85beta form similar, but distinct, insulin receptor signaling complexes.