The patch-clamp technique was used to study the effects of nystatin on a cloned delayed rectifier potassium channel (Kv1.3) expressed in Chinese hamster ovary (CHO) cells. Kv1.3 currents recorded in the whole-cell configuration, using an intracellular solution containing nystatin, were subjected to a time- and concentration-dependent reduction in their amplitude and in the time constants of apparent inactivation. Direct application of nystatin to the cytoplasmic side of excised inside-out patches inhibited Kv1.3 currents and this inhibition was immediately reversible upon washout of the drug. In contrast, currents mediated by another delayed rectifier (Kv3.1) were not affected by this drug. The concentrations for nystatin and its structural analog, amphotericin B, required to produce half maximal inhibition (IC50) of the current were estimated to be about 3 and 60 microM, respectively. The effects of nystatin on the amplitude and inactivation of Kv1.3 currents were not voltage-dependent. In inside-out patches, tetraethylammonium (TEA) produced a rapid block of Kv1.3 currents upon the onset of a voltage pulse, while the inhibition by nystatin developed slowly. When co-applied with TEA, nystatin potentiated the extent of the TEA-dependent block, and the kinetic effect of nystatin was slowed by TEA. In summary, nystatin, a compound frequently used in perforated patch recordings to preserve intracellular dialyzable components, specifically inhibited the potassium channel Kv1.3 at concentrations well below those required for perforation. The site of this inhibition may be different from that for TEA and is readily accessible from the cytoplasmic side of the membrane.