Using whole-cell patch clamp technique, we investigated the blocking effects of extracellular Ba2+ and Mg2+ on the inwardly rectifying K+ (KIR) currents of bovine pulmonary artery endothelial cells (BPAEC). The BPAEC KIR channel has recently been identified as Kir2.1 of the Kir2.0 subfamily. Block of KIR currents by Mg2+ (3-30 mM) was instantaneous, and increased with hyperpolarization slightly (Kd at -160 and 0 mV was 9.5 and 23.2 mM, respectively). The apparent fractional electrical distance (δ) of the Mg2+ binding site is calculated to be 0.07 from the outer mouth of the channel pore. Ba2+ (0.3-10 µM) time-dependently blocked the KIR currents with a much higher potency and stronger voltage-dependence (Kd at -160 and 0 mV was 1.0 and 41.6 µM, respectively). The Ba2+ binding site had a δ value of 0.34. Our data suggest that Mg2+ binds to a very superficial site of the KIR channel, while Ba2+ binds to a much deeper site, sensing much more of the membrane electric field. Thus, the BPAEC Kir2.1 appears to be pharmacologically different from the Kir2.1 reported before in bovine aortic endothelial cells (BAEC), which has 2 sites for Mg2+ block (a deep site in addition to a shallow one), and a superficial and low-sensitivity site for Ba2+ block.Key words: inwardly rectifying K+ channel, patch clamp, Ba2+, Mg2+, endothelial cells.