Two cDNAs that encode the G protein-coupled inwardly rectifying K + channel (GIRK, Kir3) of tunicate tadpoles (tunicate G protein-coupled inwardly rectifying K+ channel-A and -B; TuGIRK-A and -B) have been isolated. The deduced amino acid sequences showed ∼60% identity with the mammalian Kir3 family. Detected by whole mount in situ hybridization, both TuGIRK-A and -B were expressed similarly in the neural cells of the head and neck region from the tail bud stage to the young tadpole stage. By co-injecting cRNAs of TuGIRK-A and G protein β 1γ2 subunits (Gβγ) in Xenopus oocytes, an inwardly rectifying K+ current was expressed. In contrast, coinjection of TuGIRK-B with Gβγ did not express any current. When both TuGIRK-A and -B were coexpressed together with Gβγ, an inwardly rectifying K+ current was also detected. The properties of this current clearly differed from those of TuGIRK-A current, since it displayed a characteristic decline of the macroscopic conductance at strongly hyperpolarized potentials. TuGIRK-A/B current also differed from TuGIRK-A current in terms of the lower sensitivity to the Ba2+ block, the higher sensitivity to the Cs+ block, and the smaller single channel conductance. Taken together, we concluded that TuGIRK-A and -B form functional heteromultimeric G protein-coupled inwardly rectifying K+ channels in the neural cells of the tunicate tadpole. By introducing a mutation of Lys161 to Thr in TuGIRK-B, TuGIRK-A/B channels acquired a higher sensitivity to the Ba2+ block and a slightly lower sensitivity to the Cs+ block, and the decrease in the macroscopic conductance at hyperpolarized potentials was no longer observed. Thus, the differences in the electrophysiological properties between TuGIRK-A and TuGIRK-A/B channels were shown to be, at least partly, due to the presence of Lys161 at the external mouth of the pore of the TuGIRK-B subunit.