Continuous electrical pacing (EP) at 10 Hz of the peroneal nerve innervating fast-twitch muscles of the hindlimb in adult rabbits increases skeletal muscle concentrations of adenosine 3',5'-cyclic monophosphate (cAMP) by 3.1-fold at 10 days and increases β-adrenergic receptor (β-AR) density by 2.0-fold at 21 days. To determine whether β-AR, the α-subunit of guanine nucleotide proteins (G(sα)), or adenylyl cyclase is primarily responsible for pacing-induced increases in muscle cAMP, we measured adenylyl cyclase activity (ACA) in muscles that were electrically paced for 3 (n = 4), 10 (n = 8), and 21 (n = 8) days. EP resulted in a time-dependent increase in ACA that was 2.2 ± 0.3-fold (P < 0.005) at 21 days. EP significantly increased GTP-, 5'-guanylylimidodiphosphate-, isoproterenol-, NaF-, and forskolin-stimulated ACA, and propranolol administration to rabbits during EP did not alter pacing-induced changes in ACA. There were no changes in protein concentration, Na+-K+-adenosinetriphosphatase activity, or G(sα) with EP. Based on these studies, we conclude that EP appears to increase cAMP through mechanisms independent of the β-AR and through mechanisms that may involve alterations at the level of adenylyl cyclase.