1. We have studied the effects on ionic selectivity and gating of Kir2.1 of replacing Tyr (Y) in the GYG signature sequence with Phe (Y145F), Leu (Y145L), Met (Y145M), Ala (Y145A) or Val (Y145V). 2. The mutant Y145F showed no changes in ionic selectivity (as indicated by the permeability coefficient ratios PNa/PK or PRb/PK), indicating that a hydrogen bond between Tyr and other residues is not essential for K+ selectivity. Y145L, Y145M, Y145A and Y145V did not express as monomers. 3. None of the channels made from covalently linked tandem dimers with wild-type and mutant subunits (WT-mutant) had altered ionic selectivity (PNa/PK or PRb/PK), indicating that 4-fold symmetry is not required. 4. Macroscopic currents activated under hyperpolarization and the time constants for activation were reduced e-fold per 23 m V hyperpolarization in wild-type. This gating, believed to be due to the release of polyamines from the pore, was little affected by mutation of Y145. There was similarly little effect on the relationship between chord conductance (gK) and membrane potential. 5. Unitary conductance (140 mM [K+]o) was also little affected by mutation and was reduced only in channels formed from WT-Y145M, from 22.7 ± 0.4 pS (n = 5) in wild-type to 17.1 ± 0.5 pS (n = 4) in WT-Y145M. 6. Steady-state recording of unitary currents showed that channel open times were affected by the residue that replaced Tyr in GYG. Channel openings were particularly brief in WT-Y145V, where the mean open time was reduced from 102 ms at - 120 mV in wild-type to 6 ms in WT-Y145V. 7. Thus in Kir2.1, GFG can act as a K+ selectivity filter, as can G(L/M/A/V)G, at least in dimers also containing GYG. Channel open time duration depended on the residue at position 145, consistent with the H5 region helping to determine the dwell time of the channel in the open state.