1. Ca2+ and K+ currents were measured in single atrial and ventricular myocytes from frog heart with the whole-cell patch-clamp technique. 2. K+ currents were blocked with intra- and extracellular Cs+ and the fast Na+ current was blocked with tetrodotoxin (TTX). The Ca2+ current (ICa) was evoked by a depolarizing pulse from -80 to 0 mV. ICa was larger in ventricular (3.4 +/- 2.5 microA/cm2) than atrial (1.6 +/- 2.5 microA/cm2) myocytes. 3. Acetylcholine (ACh) had no effect on basal ICa when K+ currents were blocked with Cs+ or Ba2+. Isoprenaline increased ICa and ACh reduced the isoprenaline-stimulated current to basal levels. 4. In contrast, when K+ currents were not blocked, ACh reduced the net inward current and increased the outward current at the end of the depolarizing pulse. The outward current was studied in the presence of Cd2+ to block ICa. The steady-state current-voltage relationship inwardly rectified and reversed near the K+ reversal potential (EK). The magnitude of the steady-state ACh-activated K+ current at 0 mV was 1.0 +/- 0.7 microA/cm2 in ventricular cells and 3.67 +/- 1.7 microA/cm2 in atrial cells. 5. With depolarization, the outward current increased instantaneously and then decreased to a new steady level. The first phase of the decay occurred with a time constant similar to that of the activation of ICa. The Cd2+-sensitive current (corresponding to ICa) was obtained by subtracting currents in the presence and absence of Cd2+. The Cd2+-sensitive current was not affected by ACh. 6. The apparent effect of ACh on basal ICa can be explained quantitatively by activation of a time-dependent K+ current by ACh that contaminates ICa.