Thiazolidinediones have been shown to activate AMP-activated protein kinase activity in cultured cells. Whether they have a similar effect in vivo and if so whether it is physiologically relevant is not known. To assess these questions, we examined the effects of pioglitazone, administered orally to intact rats, on AMPK phosphorylation (AMPK-P) (a measure of its activation) and acetyl CoA carboxylase (ACC) activity and malonyl CoA concentration in rat liver and adipose tissue. In the first study, measurements were made in the Dahl-salt-sensitive rat (Dahl-S), a strain of Sprague–Dawley rat with endogenous hypertriglyceridemia and high levels of malonyl CoA that are restored to control values by pioglitazone. Treatment with pioglitazone (20mg/kg bw/day for 3 weeks) did not significantly increase either P-AMPK or P-ACC (which varies inversely with ACC activity) in control rats. However, in the Dahl-S rats values for AMPK-P and ACC-P were 50% lower than in control rats and were doubled by pioglitazone treatment. In a second study, the effects of two weeks treatment with pioglitazone (3mg/kg bw/day administered orally) were evaluated in Wistar rats. Under basal conditions (no manipulation of the animals), pioglitazone increased AMPK phosphorylation by twofold and decreased ACC activity and the concentration of malonyl CoA by 50% in liver. Following a euglycemic–hyperinsulinemic clamp (6h), 50% decreases in AMPK and ACC phosphorylation (indicating an increase in its activity) and comparable increases in malonyl CoA concentration were observed in liver and adipose tissue. In both tissues, pre-treatment with pioglitazone prevented these changes. Where studied (in Wistar rats under basal conditions) treatment with pioglitazone decreased the concentration of ATP by 1/3 and increased the concentration of ADP and AMP in liver. The results indicate that treatment with pioglitazone can increase AMPK activity in rat liver and adipose tissue in a variety of circumstances. They also suggest that this activation of AMPK may be mediated by a change in cellular energy state. Whether these effects of pioglitazone contribute to its insulin-sensitizing and other actions in vivo remains to be determined.