We have used murine erythroleukemia cells (MEL cells) to investigate the role of phosphatidylinositol 3-kinase (PI 3-kinase) in erythroid differentiation. When treated with dimethyl sulfoxide (DMSO), MEL cells grown on a fibronectin matrix become committed to erythroid differentiation asynchronously, with 90% of cells becoming committed by Day 3 of treatment. We found that during the first 3 days of DMSO treatment MEL cells showed a twofold increase in total PI 3-kinase activity and a fourfold increase in the highly phosphorylated PI 3-kinase product, PIP3. At the same time there was no change in the content of p85, the PI 3-kinase regulatory subunit. After Day 3, PI 3-kinase activity declined, in parallel with a disappearance of p85 antigen from the cells. Inclusion of the PI 3-kinase inhibitor Wortmannin in the culture medium resulted in an inhibition of cellular PI 3-kinase activity and a delay in DMSO-induced erythroid differentiation. These data suggest that PI 3-kinase may play a critical role during commitment of MEL cells to erythroid differentiation.