Ventricular and atrioventricular nodal cells from guinea pig and rabbit hearts were isolated by perfusing the heart with collagenase (Langendorff perfusion). In these cells the cyclic nucleotides cAMP and cGMP or Ca and EGTA were injected through a microelectrode by pressure (0.5-3 kg/cm2). The effect of injection on both the action potential and the hyperpolarization induced by acetylcholine was studied. The following results were obtained. 1. cAMP prolonged the ventricular action potential and shifted the plateau to more positive potentials. The configuration of the A-V nodal action potential was not detectably changed by cAMP injection, but the spontaneous rate was increased. 2. cGMP first shortened the ventricular action potential. In most experiments this effect was followed by long lasting prolongation of the action potential. 3. Both extracellular and intracellular application of dibutyryl cGMP shortened the ventricular action potential but did not produce a subsequent prolongation. However, prolongation was observed on injection of GMP, the direct metabolite. 4. Injection of cGMP in nodal cells did not hyperpolarize the membrane nor slow the spontaneous rate; rather, an increase in rate was observed. 5. The acetylcholine-induced hyperpolarization was not altered in amplitude or time course by the injection of cAMP, cGMP, Ca or EGTA. 6. The results support the hypothesis that cGMP might be involved in the control of voltage-controlled ionic channels but suggest that it does not play a role as a mediator of the classical muscarinic action i.e. the activation of a specific potassium channel by acetylcholine.