Large conductance chloride (maxi-Cl(-)) currents have been recorded in some cells, but there is still little information on the molecular nature of the channel underlying this conductance. We report here that tweety, a gene located in Drosophila flightless, has a structure similar to those of known channels and that human homologues of tweety (hTTYH1-3) are novel maxi-Cl(-) channels. hTTYH3 mRNA was found to be distributed in excitable tissues. The whole cell current of hTTYH3 was large enough to be discriminated from the control but emerged only after treatment with ionomycin. Analysis of pore mutants suggested that positively charged amino acids contributed to anion selectivity. Like a maxi-Cl(-) channel in situ, the hTTYH3 single channel showed 26-picosiemen linear current voltage, complex kinetics, 4,4'-diisothiocyanato-stilbene-2,2'-disulfonic acid sensitivity, subconductance, and the permeability order of I(-) > Br(-) > Cl(-). Similarly, hTTYH2 encoded an ionomycin-induced maxi-Cl(-) channel, but TTYH1 encoded a Ca(2+)-independent and swelling-activated maxi-Cl(-) channel. Therefore, the hTTYH family encoded maxi-Cl(-) channels of mammals. Further studies on the hTTYH family should lead to the elucidation of physiological and pathophysiological roles of novel Cl(-) channel molecules.