Cardiac inward rectifier K⁺ currents (I_K1) play an important role in maintaining resting membrane potential and contribute to late phase repolarization. Members of the K_ir2.x channel family appear to encode for I_K1. The purpose of this study was to determine the molecular composition of cardiac I_K1 in rabbit ventricle. Western blots revealed that K_ir2.1 and K_ir2.2, but not K_ir2.3, are expressed in rabbit ventricle. Culturing rabbit myocytes resulted in a ∼50 % reduction of I_K1 density after 48 or 72 h in culture which was associated with an 80 % reduction in K_ir2.1, but no change in K_ir2.2, protein expression. Dominant‐negative (DN) constructs of K_ir2.1, K_ir2.2 and K_ir2.3 were generated and tested in tsA201 cells. Adenovirus‐mediated over‐expression of K_ir2.1dn, K_ir2.2dn or K_ir2.1dn plus K_ir2.2dn in cultured rabbit ventricular myocytes reduced I_K1 density equally by 70 % 72 h post‐infection, while AdK_ir2.3dn had no effect, compared to green fluorescent protein (GFP)‐infected myocytes. Previous studies indicate that the [Ba²⁺] required for half‐maximum block (IC₅₀) differs significantly between K_ir2.1, K_ir2.2 and K_ir2.3 channels. The dependence of I_K1 on [Ba²⁺] revealed a single binding isotherm which did not change with time in culture. The IC₅₀ for block of I_K1 was also unaffected by expression of the different DN genes after 72 h in culture. Taken together, these results demonstrate functional expression of K_ir2.1 and K_ir2.2 in rabbit ventricular myocytes and suggest that macroscopic I_K1 is predominantly composed of K_ir2.1 and K_ir2.2 heterotetramers.