The contribution of Ca(2+)-activated K(+) channels to hyperpolarizing after-potentials (HAP) of action potentials, to spike-frequency adaptation and thus to the shaping of discharge pattern, was examined in rat supraoptic magnocellular neurosecretory cells. In addition, the expression of BK channels and SK3 subunits of SK channels was studied using double immunofluorescence detection. The presence of BK channels and SK3 subunits was detected in many supraoptic neurones containing either vasopressin or oxytocin. Current-clamp recordings of current-induced spike trains revealed that HAPs comprise a fast and a slow HAP (fHAP and sHAP). Correlation analyses revealed that the increase of the fHAP in amplitude and spike broadening were correlated to a moderate gradual increase of the interspike interval and thus to weak spike-frequency adaptation. By contrast, marked prolongation of the interspike interval and strong spike-frequency adaptation depended on the appearance and on the amplitude of the sHAP. The sHAP and spike-frequency adaptation were blocked by cadmium, as well as by the SK channel antagonist apamin. The fHAP was attenuated by the BK channel antagonist iberiotoxin (IbTX), by the BK/IK channel antagonist charybdotoxin (ChTX) and by apamin. ChTX attenuated fHAPs throughout the entire spike train. By contrast, the IbTX-induced attenuation of the fHAP was restricted to the initial part of the spike train, while the apamin-induced attenuation slowly increased with the progression of the spike train. These results suggest that strong spike-frequency adaptation in supraoptic neurones essentially depends on the generation of the sHAP by activation of SK channels. Comparison of effects of IbTX, ChTX and apamin suggests a complementary contribution of SK-, BK- and IK-channels to fHAPs.