Insulin resistance is a cardinal feature of normal pregnancy and excess growth hormone (GH) states, but its underlying mechanism remains enigmatic. We previously found a significant increase in the p85 regulatory subunit of phosphatidylinositol kinase (PI 3-kinase) and striking decrease in IRS-1-associated PI 3-kinase activity in the skeletal muscle of transgenic animals overexpressing human placental growth hormone. Herein, using transgenic mice bearing deletions in p85α, p85α, or insulin-like growth factor-1, we provide novel evidence suggesting that overexpression of p85α is a primary mechanism for skeletal muscle insulin resistance in response to GH. We found that the excess in total p85 was entirely accounted for by an increase in the free p85α-specific isoform. In mice with a liver-specific deletion in insulin-like growth factor-1, excess GH caused insulin resistance and an increase in skeletal muscle p85α, which was completely reversible using a GH-releasing hormone antagonist. To understand the role of p85α in GH-induced insulin resistance, we used mice bearing deletions of the genes coding for p85α or p85β, respectively (p85α+/- and p85β-/-). Wild type and p85β-/- mice developed in vivo insulin resistance and demonstrated overexpression of p85α and reduced insulin-stimulated PI 3-kinase activity in skeletal muscle in response to GH. In contrast, p85α+/- mice retained global insulin sensitivity and PI 3-kinase activity associated with reduced p85α expression. These findings demonstrated the importance of increased p85α in mediating skeletal muscle insulin resistance in response to GH and suggested a potential role for reducing p8α as a therapeutic strategy for enhancing insulin sensitivity in skeletal muscle.