Drosophila mutants provide an ideal model to study channel-type specificity of ion channel regulation in situ. In this study, the effects of divalent cations on voltage-gated K+ currents were investigated in acutely dissociated central neurons of Drosophila third instar larvae using the whole-cell patch-clamp recording. Our data showed that micromolar Cd2+ enhanced the peak inactivating current (I-A) without affecting the delayed component (I-K ). The same results were obtained in Ca2+-free external solution, and from slo(1) mutation, which eliminates transient Ca2+-activated K+ current. Micromolar d +and Zn, and millimolar Ca and Mg all shifted the steady-state inactivation curve Of IA without affecting the voltage-dependence of I-A activation, whereas millimolar Cd2+ markedly affected both the activation and steady-state inactivation curves for IA. Divalent cations affected IA with different potency; the sequence was: Zn2+ > Cd2+ > Ca2+ > Mg2+. The modulation of I-A by Cd2+ was partially inhibited in Sh(M), a null Shaker (one of I-A-encoding genes) mutation. Taken together, the channel-type specificity, the asymmetric effects on IA activation and inactivation kinetics, and the diverse potency of divalent cations all strongly support the idea that physiological divalent cations modulate A-type K+ channels through specific binding to extracellular sites of the channels.