The levels of guanosine 3',5'-cyclic phosphate (cGMP) and adenosine 3',5'-cyclic phosphate (cAMP) were measured in rat hearts after perfusion with acetylcholine to determine if parallel or independent changes occurred in the levels of these two cyclic nucleotides. It was found that after perfusion with the cholinergic agent tissue, cGMP levels increased as much as 140%. This was accompanied by no change or slight decreases in cardiac cAMP concentrations. The increases observed in cGMP levels were found to parallel the negative inotropic but not the negative chronotropic effects of acetylcholine. Perfusion with isoproterenol led to increases in the rate and force of cardiac contractility and a lowering of cGMP levels. It was concluded that the tissue concentrations of cGMP and cAMP in the perfused rat heart can vary independently and that these two tissue cyclic nucleotides probably do not share the same metabolic or functional role in this tissue.