The principal selectivity filter of K⁺ channels is found in the pore (P-) domain and contains a consensus sequence of amino acids; TxxTxGYG. However, the two P-domains of tandem pore K⁺ channels lack sequence identity. In murine TASK-1, the residues in P1 are TVITTIGYG and in P2, ITLTTIGFG. We have made substitutions of selected residues in P1 and P2. Channels were then expressed in oocytes taken from Xenopus frogs that had been anaesthetised by immersion in 0.3% w/v MS222 and killed by destruction of the brain and spinal cord. Two-electrode voltage clamp was used to measure the shift in reversal potential when K⁺ in the external medium was replaced by Rb⁺ or Na⁺.In voltage gated (Taglialatela et al., 1993) and inward rectifier (Passmore 2003) channels, the residue preceding the GYG triplet of the signature sequence is important in determining the relative permeability of the channel to Rb⁺.In TASK-1 the equivalent residue is an isoleucine (I) in both P1 and P2. We made the mutations I to S in P1 and I to V, L, S and T in P2. The mutation I94S in P1 exhibited significantly increased Rb⁺ permeability (P_Rb/P_K = 0.97 ± 0.07; n=8); compared to wild type (P_Rb/P_K = 0.77 ± 0.02; n=6; P<0.05). However mutation of I200 to S, T or V did not significantly alter Rb⁺ permeability, P_Rb/P_K being 0.81 ± 0.02 (n = 5), 0.78 ± 0.06 (n = 5) and 0.65 ± 0.04 (n = 3; P>0.05) for S, T and V mutants respectively. Only I200L showed increased permeability with respect to Rb⁺,with a P_Rb/P_K of 1.06 ± 0.05 (P<0.001 compared to wild type).We were surprised to find that all mutants showed a significant increase in permeability to Na⁺.This increase was from a P_Na/P_K of 0.02. ± 0.003 (n = 6) in wild type to 0.66 ± 0.08 (n = 8) in I94S. In mutants of P2, the increase was to 0.17 ± 0.03 (n=3), 0.38 ± 0.06 (n=5), 0.56 ± 0.02 (n=5) and 0.70 ± 0.06 (n=5) for V, L, S and T respectively, the increase in Na⁺ permeance being greater with the hydrophilic substitutions.Mutation of the equivalent residue (Val) in Kv (Shaker) channels (Heginbotham et al., 1994) to L and T gave channels that are highly selective against Na⁺. Our results show that I94 and I200 are crucial for selectivity against Na⁺ in TASK-1 with even conservative substitutions (to V and L) resulting in a loss of selectivity.