Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is a tumor suppressor gene that is frequently mutated in brain, uterine, and prostate cancers. The protein phosphatase activity is poorly defined. We demonstrate that insulin stimulates phosphorylation of tyrosine and threonine/proline residues on the p85 regulatory subunit of PI3K in Huh-7, and HEK 293 cells. The specificity of PTEN binding and dephosphorylation of PI3K appears to reside on the p8513 subunit. Therefore, the PTEN phosphatase is active against the PI3K p8513 subunit and dephosphorylates a protein involved in insulin signaling where known downstream consequences are increased cell migration, motility, and invasion. (C) 2010 Elsevier Inc. All rights reserved.