At concentrations around 10^-9 M or higher, glucagon increases cardiac contractility by activating adenylate cyclase/cyclic adenosine monophosphate (AC/cAMP). However, blood levels in vivo, in rats or humans, rarely exceed 10_-10 M. We investigated whether physiologic concentrations of glucagon, not sufficient to increase contractility or ventricular cAMP levels, can influence fuel metabolism in perfused working rat hearts. Two distinct glucagon dose-response curves emerged. One was an expected increase in left ventricular pressure (LVP), occurring between 10^-9.5 and 10^-8 M. The elevations in both LVP and ventricular cAMP levels produced by the maximal concentration (10^-8 M) were blocked by the AC inhibitor NKY 80 (20 µM). The other curve, generated at much lower glucagon concentrations and overlapping normal blood levels (10^-11 - 10^-10 M), consisted of a dose-dependent and marked stimulation of glycolysis with no change in LVP. In addition to stimulating glycolysis, glucagon (10^-10 M) also increased glucose oxidation and suppressed palmitate oxidation - mimicking known effects of insulin - without increasing ventricular cAMP levels. Elevations in glycolytic flux produced by either glucagon (10^-10 M) or insulin (4 X 10^-10 M) were abolished by the phosphoinositide 3-kinase (PI3-K) inhibitor LY294002 (10 µM) but not significantly affected by NKY 80. Glucagon also, like insulin, enhanced the phosphorylation of Akt/PKB, a downstream target of PI3-K, and these effects were also abolished by LY294002. The results are consistent with the hypothesis that physiologic levels of glucagon produce insulin-like increases in cardiac glucose utilization in vivo through activation of PI3-K and not AC/cAMP.