The purpose of the present study was to examine how apamin interacts with the three cloned subtypes of small-conductance Ca2+-activated K+ channels (hSK1, rSK2 and rSK3). Expression of the SK channel subtypes in Xenopus laevis oocytes resulted in large outward currents (0.5-5 microA) after direct injection of Ca2+. In all three cases the Ca2+-activated K+ currents could be totally inhibited by 500 nM apamin. Dose-response curves revealed a subtype-specific affinity for the apamin-induced inhibition with IC50 values of 704 pM and 196 nM (biphasic) for hSK1, 27 pM for rSK2 and 4 nM for rSK3. Consistent with these results, membranes prepared from oocytes expressing the SK channel subtypes bound 125I-labelled apamin with distinct dissociation constants (Kd values) of approx. 390 pM for hSK1, 4 pM for rSK2 and 11 pM for rSK3. These results show that apamin binds to and blocks all three subtypes of cloned SK channels, and the distinct values for IC50 and Kd suggest that apamin may be useful for determining the expression pattern of SK channel subtypes in native tissue.