1. A low stringency polymerase chain reaction (PCR) homology screening procedure was used to probe a mouse liver cDNA library to identify novel inward rectifier K+ channel genes. A single gene (mLV1) was identified that exhibited extensive sequence homology with previously cloned inward rectifier K+ channel genes. The mLV1 gene showed greatest sequence identity with genes belonging to the Kir4 subfamily. The amino acid sequence of mLV1 was 96% identical to a Kir channel cloned from human kidney (hKir4.2), and ~60% identical to the Kir4.1 channel cloned from human and rat, so that mLV1 was classified as mKir4.2. 2. Xenopus oocytes injected with cRNA encoding mKir4.2 displayed a large inwardly rectifying K+ current, while control oocytes injected with H2O displayed no similar K+ current. The current was blocked by Ba2+ and Cs+ in a voltage-dependent fashion and displayed inward rectification that was intermediate between that of the strong inward rectifier Kir2.1. and the weak inward rectifier Kir1.1. The current was weakly blocked by TEA in a voltage-independent fashion. 3. mKir4.2 current was subject to modulation by several distinct mechanisms. Intracellular acidification decreased mKir4.2 current in a reversible fashion, while activation of protein kinase C decreased mKir4.2 current in a manner that was not rapidly reversible. Incubation of oocytes in elevated [K+] produced a slowly developing enhancement of current. 4. Oocytes co-injected with cRNA for mKir4.2 and Kir5.1, a protein that does not form functional homomeric channels, displayed membrane currents with properties distinct from those expressing mKir4.2 alone. Co-injected oocytes displayed larger currents than mKir4.2, with novel kinetic properties and an increased sensitivity to Ba2+ block at negative potentials, suggesting that mKir4.2 forms functional heteromultimeric channels with Kir5.1, as has been shown for Kir4.1. 5. These results demonstrate for the first time that a Kir4.2 channel gene product forms functional channels in Xenopus oocytes, that these Kir channels display novel properties, and that Kir4.2 subunits may be responsible for physiological modulation of functional Kir channels.