We have examined the effects of certain mutations of the selectivity filter and of the membrane helix M2 on Ba2＋ blockage of the inward rectifier potassium channel, Kir 2.1. We expressed mutant and wild type murine Kir 2.1 in Chinese hamster ovary (CHO) cells and used the whole cell patch-clamp technique to record K＋ currents in the absence and presence of externally applied Ba2＋. Wild type Kir2.1 was blocked by externally applied Ba2＋ in a voltage and concentration dependent manner. Mutants of Y145 in the selectivity filter showed little change in the kinetics of Ba2＋ blockage. The estimated Kd(0) was 108ՌM for Kir2.1 wild type, 124ՌM for a concatameric WT-Y145V dimer, 109ՌM for a WT-Y145L dimer, and 267ՌM for Y145F. Mutant channels T141A and S165L exhibit a reduced affinity together with a large reduction in the rate of blockage. In S165L, blockage proceeds with a double exponential time course, suggestive of more than one blocking site. The double mutation T141A/S165L dramatically reduced affinity for Ba2＋, also showing two components with very different time courses. Mutants D172K and D172R (lining the central, aqueous cavity of the channel) showed both a decreased affinity to Ba2＋ and a decrease in the on transition rate constant (kon). These results imply that residues stabilising the cytoplasmic end of the selectivity filter (T141, S165) and in the central cavity (D172) are major determinants of high affinity Ba2＋ blockage in Kir 2.1.