Calcium-activated potassium current was studied in cultured rabbit retinal pigment epithelial (RPE) cells using whole-cell and single channel patch-clamp recording techniques. When K+was the principal cation in the electrode, depolarizing voltage steps from a holding potential of–60 mV activated outwardly rectifying current. Outward K+current was increased by the Ca2+ionophore ionomycin and reduced when the extracellular Ca2+concentration was decreased from 2.5 mM to 100 nM in the presence of ionomycin. Outward K+current recorded in the presence of ionomycin was blocked by iberiotoxin and by charybdotoxin. Single channel recording from cell-attached and excised membrane patches revealed a large conductance Ca2+-activated K+(K(Ca)) channel. Identification of K(Ca)channels was based on: 1) the voltage-dependence of channel opening; 2) the large unitary conductance (>200 pS with symmetrical 130 mM K+); 3) the dependence of the reversal potential on the K+gradient; and 4) increased channel opening after exposure of the cytosolic surface of excised membrane patches to elevated Ca2+. These results demonstrate that Ca2+-activated K+channels are present in rabbit RPE cells and may play an essential role in the regulation of membrane potential and ion transport.