We studied the K(+)-selective conductances in primary cultures of rat renal inner medullary collecting duct (IMCD) using perforated-patch and conventional whole cell techniques. Depolarizations above -20 mV induced a time-dependent outward K(+) current (I(vto)) similar to a delayed rectifier. I(vto) showed a half-maximal activation around 5.6 mV with a slope factor of 6.8 mV. Its K(+)/Na(+) selectivity ratio was 11.7. It was inhibited by tetraethylammonium, quinidine, 4-aminopyridine, and Ba(2+) and was not Ca(2+) dependent. The delayed rectifying characteristics of I(vto) prompted us to screen the expression of Kv1 and Kv3 families by RT-PCR. Analysis of RNA isolated from cell cultures revealed the presence of three Kv alpha-subunits (Kv1.1, Kv1.3, and Kv1.6). Western blot analysis with Kv alpha-subunit antibodies for Kv1.1 and Kv1.3 showed labeling of approximately 70-kDa proteins from inner medulla plasmatic and microsome membranes. Immunocytochemical analysis of cell culture and kidney inner medulla showed that Kv1.3 is colocalized with the Na(+)-K(+)-ATPase at the basolateral membrane, although it is also in the cytoplasm. This is the first evidence of recording, protein expression, and localization of a voltage-gated Kv1 in the kidney IMCD cells.