Wortmannin, an inhibitor of PI3-kinase, was systemically infused during a hyperinsulinemic euglycemic clamp to investigate its effects in vivo. Rats were infused under anesthesia with saline, 10 or 20mU·min^-1·kg^-1 insulin, wortmannin (1µg·min^-1·kg^-1) + saline, or wortmannin + insulin (10mU·min^-1·kg^-1); wortmannin was present for 1h before and throughout the 2h clamp. Femoral blood flow (FBF), glucose infusion rate to maintain euglycemia (GIR), glucose appearance (Ra), glucose disappearance (Rd), capillary recruitment by 1-methylxanthine metabolism (MXD), hind leg glucose uptake (HLGU), liver, muscle and aorta Akt phosphorylation (P-Akt/Akt) and plasma insulin concentrations (PI) were determined. PI increased from 410 ± 49 to 1680 ± 430pM and 5060 ± 230pM with 10 and 20mU·min^-1·kg^-1 insulin, respectively. Insulin (10 and 20mU·min^-1·kg^-1) increased FBF, MXD, GIR, Rd, HLGU as well as liver, muscle and aorta P-Akt/Akt, and decreased Ra (all P < 0.05). Wortmannin alone increased PI to 5450 ± 770pM and increased Ra, Rd, HLGU, and muscle P-Akt/Akt without effect on blood glucose, FBF, MXD liver or aorta P-Akt/Akt. Wortmannin blocked FBF, MXD and liver P-Akt/Akt increases from 10mU·min^-1·kg^-1 insulin. Comparison of wortmannin + 10mU·min^-1·kg^-1 insulin and 20mU·min^-1·kg^-1 insulin alone (both at ~5,000pM PI), showed that wortmannin fully blocked the changes in FBF and Ra, and partly those of GIR, Ra, Rd, HGLU and muscle P-AKT/Akt. In summary, wortmannin in vivo increases plasma insulin, fully inhibits insulin-mediated effects in liver and aorta and partially those of muscle, where the latter may result from inhibition of insulin-mediated increases in blood flow and capillary recruitment.