The gut hormone, glucagon-like peptide-1 (GLP-1), which is secreted in nanomolar amounts in response to nutrients in the intestinal lumen, exerts cAMP/protein kinase A–mediated insulinotropic actions in target endocrine tissues, but its actions in heart cells are unknown. GLP-1 (10 nmol/L) increased intracellular cAMP (from 5.7±0.5 to 13.1±0.12 pmol/mg protein) in rat cardiac myocytes. The effects of cAMP-doubling concentrations of both GLP-1 and isoproterenol (ISO, 10 nmol/L) on contraction amplitude, intracellular Ca²⁺ transient (CaT), and pH_i in indo-1 and seminaphthorhodafluor (SNARF)–1 loaded myocytes were compared. Whereas ISO caused a characteristic increase (above baseline) in contraction amplitude (160±34%) and CaT (70±5%), GLP-1 induced a significant decrease in contraction amplitude (−27±5%) with no change in the CaT after 20 minutes. Neither pertussis toxin treatment nor exposure to the cGMP-stimulated phosphodiesterase (PDE2) inhibitor erythro-9-(2-hydroxy-3-nonyl)adenine or the nonselective PDE inhibitor 3-isobutyl-1-methylxanthine nor the phosphatase inhibitors okadaic acid or calyculin A unmasked an ISO-mimicking response of GLP-1. In SNARF-1–loaded myocytes, however, both ISO and GLP-1 caused an intracellular acidosis (ΔpH_i −0.09±0.02 and −0.08±0.03, respectively). The specific GLP-1 antagonist exendin 9-39 and the cAMP inhibitory analog Rp-8CPT-cAMPS inhibited both the GLP-1–induced intracellular acidosis and the negative contractile effect. We conclude that in contrast to β-adrenergic signaling, GLP-1 increases cAMP but fails to augment contraction, suggesting the existence of functionally distinct adenylyl cyclase/cAMP/protein kinase A compartments, possibly determined by unique receptor signaling microdomains that are not controlled by pertussis toxin–sensitive G proteins or by enhanced local PDE or phosphatase activation. Furthermore, GLP-1 elicits a cAMP-dependent modest negative inotropic effect produced by a decrease in myofilament-Ca²⁺ responsiveness probably resulting from intracellular acidification.