Calcium-activated chloride currents (I(Cl(Ca))) can be recorded in almost all cells, but the molecular identity of the channels underlying this Cl- conductance is still incompletely understood. Here, I report that tweety, a gene located in Drosophila flightless, possesses five or six transmembrane segments, and that a human homologue of tweety (hTTYH3) is a novel large-conductance Ca2+-activated Cl- channel, while the related gene, hTTYH1, is a swelling-activated Cl- current. hTTYH3 is expressed in excitable tissues, including the heart, brain and skeletal muscle, whereas hTTYH1 is expressed mainly in the brain. Expression of hTTYH3 in CHO cells generated a unique Cl- current activated by an increase in the intracellular Ca2+ concentration. The hTTYH3-induced Cl- current had a linear current-voltage (I-V) relationship, a large single-channel conductance (260 pS) and the anion permeability sequence I- > Br- > Cl-. Like native Ca2+-activated Cl- channels, the hTTYH3 channel showed complex gating kinetics and voltage-dependent inactivation, and was dependent on micromolar intracellular Ca2+ concentration. Expression in CHO cells of an hTTYH1 splice variant that lacks the C-terminal glutamate-rich domain of hTTYH1 (hTTYH1sv) generated a swelling-activated Cl- current. I conclude that investigation of the tweety family will provide important information about large-conductance Cl- channel molecules.