Insulin stimulation increases cell proliferation and energy metabolism by activating the insulin receptor substrate I (IRS-1)-signaling pathways. This downstream signaling is mediated by interactions of specific tyrosyl phosphorylated (PY) IRS-1 motifs with SH2-containing molecules such as growth-factor receptor-bound protein 2 (Grb2) and Syp. Ethanol inhibits insulin-stimulated tyrosyl phosphorylation of IRS-1 and DNA synthesis. This study explores the roles of the Grb2- and Syp-binding motifs of IRS-1 in relation to the inhibitory effects of ethanol on insulin-stimulated DNA synthesis, proliferating cell nuclear antigen (PCNA) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression, and activation of mitogen-activated protein kinase (MAPK), which is known to be essential for cell proliferation. NIH3T3 cells were stably transfected with wild-type IRS-1, or IRS-1 mutated at the Grb2 (IRS-1DeltaGrb2), Syp (IRS-1DeltaSyp), or Grb2 and Syp (IRS-1DeltaGrb2DeltaSyp)- binding sites. Cells transfected with IRS-1 had increased levels of DNA synthesis, PCNA, GAPDH, and activated MAPK. The IRS-1DeltaGrb2 transfectants were highly responsive to insulin stimulation, achieving levels of GAPDH, PCNA, and activated MAPK that were higher than control. In contrast, the IRS-1DeltaSyp and IRS-1DeltaGrb2DeltaSyp transfectants had reduced levels of DNA synthesis, PCNA, and activated MAPK. Ethanol exposure decreased insulin-stimulated DNA synthesis, PCNA, GAPDH, and activated MAPK levels in all clones, but the wild-type IRS-1 transfectants were relatively resistant, and the IRS-1DeltaGrb2 transfectants were extraordinarily sensitive to these inhibitory effects of ethanol. The findings suggest that insulin-stimulated DNA synthesis and PCNA expression are mediated through the Syp-binding domain, whereas GAPDH expression and MAPK activation are modulated through both the Grb2 and Syp motifs of IRS-1. In addition, ethanol exposure may preferentially inhibit downstream signaling that requires interaction between Syp and PY-IRS-1.