1. Properties and functions of Na(+)-activated K+ (KNa) channels in the soma of motoneurones were studied in spinal cord slices of newborn rat. KNa channels had a conductance of 44.8 pS in 5.6 mM external K+ (Ko+)/106 mM internal K+ (Ki+) solutions and 139.2 pS in 155 mM Ko+/85 mM Ki+ solutions. KNa channels were voltage independent and needed a relatively high [Na+]i to become active (EC50 = 39.9 mM). Li+ could not substitute for Na+ in activation of KNa channels. The channels were predominantly found in the vicinity of cell processes, in the regions of most probable accumulation of cytoplasmic Na+. 2. In current-clamp experiments, the shape of the single action potential (AP) recorded in Ca(2+)-free Ringer solution was not changed after substitution of external Na+ with Li+. However, 0.4-0.8 s trains of APs were followed by a slow (1-2s) after-hyperpolarization (sAHP), which reversibly disappeared when external Na+ was replaced by Li+. Na(+)-activated sAHP persisted after addition of ouabain and its amplitude was even increased in K(+)-free Ringer solution. sAHP disappeared when the membrane potential was equal to the K+ equilibrium potential. This indicated that sAHP resulted from activation of a Na(+)-dependent K+ conductance, rather than from activation of the electrogenic Na(+)-K+ pump. 3. In conclusion, KNa channels can play an important role in excitability of motoneurones. KNa channels do not make a contribution to the single AP, but they can be activated by a local accumulation of internal Na+ during trains of APs. A Na(+)-activated K+ conductance can reduce membrane excitability and contribute to regulation of AP firing in motoneurones.