AMP-activated protein kinase (AMPK) is known to be activated by phosphorylation on Thr172 in response to an increased AMP/ATP ratio. We report here that such an activation indeed occurred in anaerobic rat hearts and that it was antagonized (40-50%) when the hearts were pre-treated with 100 nM insulin. The effect of insulin (1) was blocked by wortmannin, an inhibitor of phosphatidylinositol-3-kinase; (2) only occurred when insulin was added before anoxia, suggesting a hierarchical control; (3) resulted in a decreased phosphorylation state of Thr172 in AMPK and (4) was unrelated to changes in the AMP/ATP ratio. This is the first demonstration that AMPK activity could be changed without a detectable change in the AMP/ATP ratio of the cardiac cell.