Small conductance Ca(2+)-activated K(+) channels (SK) regulate action potential (AP) firing properties and excitability in many central neurons. However, the functional roles of SK channels of parasympathetic cardiac motoneurons (PCMNs) in the nucleus ambiguus have not yet been well characterized. In this study, the tracer X-rhodamine-5 (and 6)-isothiocyanate (XRITC) was injected into the pericardial sac to retrogradely label PCMNs in FVB mice at postnatal days 7-9. Two days later, XRITC-labeled PCMNs in brain stem slices were identified. With the use of whole cell current clamp, single APs and spike trains of different frequencies were evoked by current injections. We found that 1) PCMNs have two different firing patterns: the majority of PCMNs (90%) exhibited spike frequency adaptation (SFA) and the rest (10%) showed less or no adaptation; 2) application of the specific SK channel blocker apamin significantly increased spike half-width in single APs and trains and reduced the spike frequency-dependent AP broadening in trains; 3) SK channel blockade suppressed afterhyperpolarization (AHP) amplitude following single APs and trains and abolished spike-frequency dependence of AHP in trains; and 4) SK channel blockade increased the spike frequency but did not alter the pattern of SFA. Using whole cell voltage clamp, we measured outward currents and afterhyperpolarization current (I(AHP)). SK channel blockade revealed that SK-mediated outward currents had both transient and persistent components. After bath application of apamin and Ca(2+)-free solution, we found that apamin-sensitive and Ca(2+)-sensitive I(AHP) were comparable, confirming that SK channels may contribute to a major portion of Ca(2+)-activated K(+) channel-mediated I(AHP). These results suggest that PCMNs have SK channels that significantly regulate AP repolarization, AHP, and spike frequency but do not affect SFA. We conclude that activation of SK channels underlies one of the mechanisms for negative control of PCMN excitability.