1. The structural determinants of a G protein-activated inwardly rectifying potassium channel, GIRK1 (K(IR) 3.1), involved in voltage- and time-dependent gating properties were investigated by heterologous expression of chimeric constructs and point mutants in Xenopus oocytes. 2. Chimeras between GIRK1 and the weakly rectifying potassium channel, ROMK1 (K(IR)1.1), indicate that residues in the putative transmembrane segments TM1 and TM2 affect the steep inward rectification of GIRK1, while residues in the main pore-forming domain, the P-region segment, are critical for the manifestation of GIRK1 time-dependent activation. 3. Phenylalanine 137 in the P-region of GIRK1 is unique; in ROMK1, as in other inward rectifiers, there is a serine residue at this position. Mutation of the phenylalanine 137 to serine leads to expression of currents with nearly time-independent activation. 4. An acidic residue (aspartate) in TM2 partially controls the time- and voltage-dependent gating in IRK1 (K(IR)2.1). Mutation of the equivalent aspartate 173 to glutamine in GIRK1. did not abolish the time-dependent activation but did decrease the degree of inward rectification. 5. These results reveal an important role for the P-region in controlling the time-dependent gating of an inwardly recitfying potassium channel and suggest a close relationship between permeation and gating in this family of K+ channels.