Because insulin-like growth factor-I (IGF-I), insulin, and interleukin- 4 (IL-4) have known biological effects in breast cancer cells and signal through insulin-receptor substrate (IRS) adaptor proteins, we examined the expression and function of IRS-1 and IRS-2 in breast tumors and cell lines. IRS-1 and IRS-2 were expressed by cell lines and primary breast tumor specimens. IGF-I, insulin, and IL-4 treatment of MCF-7 and ZR-75, and IGF-I treatment of T47-D breast cancer cells, resulted in much greater tyrosine phosphorylation of IRS-1 compared with IRS-2. Furthermore, IGF-I stimulated greater tyrosine phosphorylation of IRS-1 than either insulin or IL-4. IGF-I treatment also enhanced association of the p85 regulatory subunit of phosphatidylinositol 3-kinase with IRS-1 and stimulated increased enzymatic activity compared with IL-4 and insulin in all three cell lines. Similarly, mitogen-activated protein kinase activity was greater in IGF-I-stimulated cells. To determine the functional significance of the activation of these pathways, we inhibited activation of phosphatidylinositol 3-kinase with wortmannin and mitogen-activated protein kinase with PD098059. Both compounds inhibited IGF-stimulated growth, suggesting that both pathways contributed to the mitogenic response to IGF-I. We conclude that IRS-1, and not IRS-2, is the predominant signaling molecule activated by IGF-I, insulin, and IL-4. Furthermore, enhanced tyrosine phosphorylation of IRS-1 by IGF-I, compared with either insulin or IL-4, is associated with greater activation of mitogenic downstream signaling pathways resulting in enhanced cell growth.