Using electrophysiological and molecular techniques, we investigated the molecular nature of an inwardly rectifying K+ channel in bovine parotid acinar (BPA) cells and examined its role in setting resting membrane potential. In whole-cell recordings from freshly isolated BPA cells, a predominant current was a K+ current rectified strongly in the inward direction. An inward conductance of the inwardly rectifying K+ (Kir) current was proportional to [K+]o(0.57). The selectivity sequence based on permeability ratios was K+ (1.00) > Rb+ (0.63) >> Li+ (0.04) = Na+ (0.02) and the sequence based on conductance ratios was K+ (1.00) >> Rb+ (0.03) = Li+ (0.03) = Na+ (0.02). The current was blocked by extracellular Ba2+ and Cs+ in a voltage- and a concentration-dependent manner, with a Kd at 0 mV of 11.6 microM and 121 mM, respectively. Cell-attached patch measurements identified 27 pS K+ channels as being the most likely to mediate whole-cell Kir currents. Addition of Ba2+ (100 microM) to the bathing solution reversibly depolarized the resting membrane potential in intact unstimulated cells. RT-PCR of RNA from bovine parotid cells revealed transcripts of bovine Kir2.1 (bKir2.1). HEK293 cells stably expressing bKir2.1 cloned from bovine parotid exhibited whole-cell and single channel Kir currents, of which electrophysiological characteristics were quantitatively similar to those of native Kir currents. Immunohistochemical studies showed a bKir2.1 immunoreactivity in BPA cells. Collectively, these results suggest that Kir2.1 may mediate native Kir currents responsible for setting resting membrane potential in BPA cells and might be, at least in part, involved in spontaneous secretion in ruminant parotid glands.