Exercise induces a decline in liver malonyl-CoA, an inhibitor of carnitine palmitoyltransferase-1. The purpose of these experiments was to determine whether this decrease in malonyl-CoA is accompanied by an activation of AMP-activated protein kinase (AMPK) and inactivation of acetyl-CoA carboxylase (ACC). Rats were killed at rest, after 10 min of running at 32 m/min up a 15% grade or at 0, 15, or 60 min postexercise after 120 min of running at 16 m/min. There was no significant difference in AMPK and ACC activities after 120 min of exercise, although a trend toward a decrease in ACC and an increase in AMPK was noted 15 min postexercise. After 10 min at 32 m/min, however, maximal ACC activity decreased from 487 ± 27 to 280 ± 39 nmol ⋅ g⁻¹ ⋅ min⁻¹, and the activation constant for citrate activation of ACC increased from 5.9 to 12.5 mM. AMPK activity increased from a resting value of 4.7 ± 0.4 to 9.8 ± 2.0 pmol ⋅ mg⁻¹ ⋅ min⁻¹after exercise. These data provide indirect evidence of phosphorylation and inactivation of liver ACC during heavy exercise. In contrast, the decrease in malonyl-CoA during long-term, low-intensity exercise may occur by mechanisms other than phosphorylation of ACC.