Activated insulin receptor (IR) interacts with its substrates, IRS-1, IRS-2, and Shc via the NPXY motif centered at Y960. This interaction is important for IRS-1 phosphorylation. Studies using the yeast two-hybrid system and sequence identity analysis between IRS-1 and IRS-2 have identified two putative elements, the PTB and SAIN domains, between amino acids 108 and 516 of IRS-1 that are sufficient for receptor interaction. However, their precise function in mediating insulin’s bioeffects is not understood. We expressed the PTB and SAIN domains of IRS-1 in HIRcB fibroblasts and 3T3-L1 adipocytes utilizing replication-defective adenoviral infection to investigate their role in insulin signalling. In both cell types, overexpression of either the PTB or the SAIN protein caused a significant decrease in insulin-induced tyrosine phosphorylation of IRS-1 and Shc proteins, IRS-1-associated phosphatidylinositol 3-kinase (PI 3-K) enzymatic activity, p70 s6k activation, and p44 and p42 mitogen-activated protein kinase (MAPK) phosphorylation. However, epidermal growth factor-induced Shc and MAPK phosphorylation was unaffected by the overexpressed proteins. These findings were associated with a complete inhibition of insulin-stimulated cell cycle progression. In 3T3-L1 adipocytes, PTB or SAIN expression extinguished IRS-1 phosphorylation with a corresponding 90% decrease in IRS-1-associated PI 3-K activity. p70 s6k is a downstream target of PI 3-K, and insulin-stimulated p70 s6k was inhibited by PTB or SAIN expression. Interestingly, overexpression of either PTB or SAIN protein did not affect insulin-induced AKT activation or insulin-stimulated 2-deoxyglucose transport, even though both of these bioeffects are inhibited by wortmannin. Thus, interference with the IRS–1-IR interaction inhibits insulin-stimulated IRS-1 and Shc phosphorylation, PI 3-K enzymatic activity, p70 s6k activation, MAPK phosphorylation and cell cycle progression. In 3T3-L1 adipocytes, interference with the IR–IRS-1 interaction did not cause inhibition of insulin-stimulated AKT activation or glucose transport. These results indicate a bifurcation or subcompartmentalization of the insulin signalling pathway whereby some targets of PI 3-K (ie, p70 s6k) are dependent on IRS–1-associated PI 3-K and other targets (ie, AKT and glucose transport) are not. IR-IRS-1 interaction is not essential for insulin’s effect on glucose transport, and alternate, or redundant, pathways exist in these cells.