We have used two experimental approaches to examine the possible role of phosphatidylinositol 3-kinase (PI 3-kinase) in the regulation of glucose transport in oocytes isolated from Xenopus laevis. Incubation of oocytes with the PI 3-kinase inhibitor wortmannin inhibited insulin-like growth factor-1-stimulated deoxyglucose uptake. Half-maximal inhibition was observed at concentrations approximately 20 nM. Conversely, we also examined the effects of microinjection of synthetic peptides designed to interact with Src homology 2 domains of the regulatory subunit of PI 3-kinase on deoxyglucose transport in oocytes. We show that a bifunctional synthetic peptide containing two YMXM consensus sequences for binding to SH2 domains stimulated both PI 3-kinase activity and deoxyglucose transport when both tyrosine residues were phosphorylated. However, non-phosphorylated or bisphosphonotyrosine peptides with the identical amino acid sequence failed to stimulate transport or PI 3-kinase activity. Taken together, these data argue strongly for a role for PI 3-kinase in the regulation of glucose transport in oocytes.