Aquaporin 6 (AQP6) is unique among mammalian AQPs in being an anion channel with negligible water permeability. However, the point mutation Asn60Gly converts AQP6 from an anion channel into a water channel. In the present study of human AQP5, we mutated Leu51 (corresponding to residue 61 in AQP6), the side chain of which faces the central pore. We evaluated function in Xenopus oocytes by two-electrode voltage clamp, video measurements of osmotic H₂O permeability (P_f), microelectrode measurements of surface pH (pHS) to assess CO₂ permeability, and surface biotinylation. We found that AQP5-L51R does not exhibit the H₂O or CO₂ permeability of the wild-type protein but instead has a novel pCMBS-sensitive current. The double mutant AQP5-L51R/C182S renders the conductance insensitive to pCMBS, demonstrating that the current is intrinsic to AQP5. AQP5-L51R has the anion permeability sequence I^– > NO₃^- NO₂^- > Br^– > Cl^– > HCO₃^- > Gluconate. Of other L51 mutants, L51T (polar uncharged) and L51V (nonpolar) retain H₂O and CO₂ permeability and do not exhibit anion conductance. L51D and L51E (negatively charged) have no H₂O or CO₂ permeability. L51K (positively charged) has an intermediate H₂O and CO₂ permeabilities and anion conductance. L51H is unusual in having a relatively low CO₂ permeability and anion conductance, but a moderate P_f. Thus, positively charged mutations of L51 can convert AQP5 from a H₂O/CO₂ channel into an anion channel. However, the paradoxical effect of L51H is consistent with the hypothesis that CO₂, in part, takes a pathway different from H₂O through AQP5.