The second transmembrane domain (TM2) of P2X receptor may form part of the ion permeation pathway. Polar amino acid side chains are conserved in TM2. We tested their participation in permeation by single-channel and whole-cell recordings, applying ATP (0.1 – 30 μM) to HEK293 cells expressing wild type (wt) and mutated receptors. Unitary conductance (γ) in outside-out patches of the wt channels was 21.7 ± 0.8 pS (n = 15; -100 mV). T330 is situated near the outer end of TM2; substitution with Ala, Cys, Gly, Ser, Trp and Tyr did not change unitary currents whereas Asp, Glu, Lys and Arg gave no detectable current. N333 could be replaced by Ala, Cys, Asp, Gly, Ser and Thr without changing γ, whereas large (Trp and Tyr) or charged (Asp, Glu, Lys, Arg) side chains reduced γ. T336 may be in the permeation pathway (Rassendren, F. et al. 1997): substitution with Cys, Glu, Asp, Gly, and Ser did not change γ, but γ was significantly reduced with Ala, Asn, Gln or Trp at this position. For T336C, ethyltrimethlammonium methane thiosulphonate reduced the probability of opening of single channels but did not change γ. T339 was less tolerant of substitution: T339S and T339C had γ similar to wt values, but T339A, T339E, T339G, T339N, and T339Q had significantly lower γ. The neighbouring residue S340 was also intolerant of substitution, with only S340E similar to wt. γ for S340A, S340G, S340K, S340N, S340Q, S340T ranged from 14 to 11 pS, whereas S340Y and S340W were both 8.6 pA/pF) and in these cells application of ATP (100 μM) evoked no further current. These results are consistent with the view (Egan T.M. et al., 2006) that the region around T339 and S340 may provide the selectivity filter. The spontaneous activity observed indicates that the entire region from N333 to S340 is also important for gating transitions, given that relatively minor disturbances to side chains in this region (eg. T339S) can greatly destabilise channel closed states.