The presence of phosphatidylinositol 3-kinase (PI 3-kinase) in mammalian skeletal muscle and its response to insulin stimulation were investigated. PI kinase, immunoprecipitated from rat soleus muscle with antibodies directed toward its 85-kDa subunit phosphorylated PI, phosphatidylinositol 4-phosphate [PI(4)P], and phosphatidylinositol 4,5,-bisphosphate [PI(4,5)P2] to yield phosphatidylinositol 3-phosphate [PI(3)P], phosphatidylinositol 3,4,-bisphosphate, and phosphatidylinositol trisphosphate in vitro. PI 3-kinase activity was also immunoprecipitated with antiphosphotyrosine [alpha-Tyr(P)] antibodies and with antibodies raised against IRS-1, a substrate of the insulin receptor protein tyrosine kinase that associates with and activates PI 3-kinase. Incubation of the soleus with insulin in vitro, or injection of insulin into rats in vivo, produced three- to fivefold increases in alpha-Tyr(P)- and alpha-IRS-1-immunoprecipitable PI 3-kinase activity. In nonstimulated soleus muscle, PI 3-kinase activity immunoprecipitated with alpha-IRS-1 or with alpha-Tyr(P) antibodies was evenly distributed between particulate (200,000-g pellet) and soluble fractions. Insulin treatment increased immunoprecipitable PI 5-kinase activity in both fractions, but the increase in alpha-Tyr-(P)-precipitable activity was greater in the particulate fraction, whereas the increase in alpha-IRS-1-precipitable activity was greater in the soluble fraction. In intact soleus muscles incubated with 32PO4, insulin increased the labeling of PI(3)P but did not affect the labeling of PI(4)P or PI(4,5)P2. Activation of PI 3-kinase by insulin was unaffected by prior denervation of the muscle, a manipulation that has been shown to cause both insulin resistance and hypersensitivity in muscles, depending on the parameter measured.(ABSTRACT TRUNCATED AT 250 WORDS)