Vascular endothelial growth factor (VEGF) isoforms exert their biological effects through receptors that possess intrinsic tyrosine kinase activity. Whether VEGF binding to its receptors recruits insulin receptor substrate (IRS) family of docking proteins to the receptor is not known. Following incubation of mouse kidney proximal tubular epithelial cells with VEGF, we observed an increase in tyrosine phosphorylation of several proteins, including one of approximately 200 kDa, suggesting possible regulation of phosphorylation of IRS proteins. VEGF augmented tyrosine phosphorylation of IRS-1 in kidney epithelial cells and rat heart endothelial cells in a time-dependent manner. In the epithelial cells, association of IRS-1 with type 2 VEGF receptor was promoted by VEGF. VEGF also increased association of IRS-1 with the p85 regulatory subunit of phosphoinositide 3-kinase (PI 3-kinase), and PI 3-kinase activity in IRS-1 immunoprecipitates was increased in VEGF-treated cells. Incubation of epithelial cells with antisense IRS-1 oligonucleotide, but not sense oligonucleotide, reduced expression of the protein and VEGF-induced PI 3-kinase activity in IRS-1 immunoprecipitates. Additionally, VEGF-induced protein synthesis was also impaired by antisense but not sense IRS-1 oligonucleotide. These data provide the first evidence that binding of VEGF to its type 2 receptor promotes association of IRS-1 with the receptor complex. This association may account for some of the increase in VEGF-induced PI 3-kinase activity, and the increase in de novo protein synthesis seen in renal epithelial cells.