In mammals, the superfamily of "Cys loop," ligand-gated ion channels (LGICs), is assembled from a pool of more than 40 homologous subunits. These subunits have been classified into four families representing channels that are gated by acetylcholine, serotonin, γ-aminobutyric acid, or glycine. By searching anonymous genomic sequence data for exons that encode characteristic motifs of the channel subunits, we have identified a novel LGIC that defines a fifth family member. Putative exons were used to isolate a full-length cDNA that encodes a protein of 411 amino acid residues. This protein (ZAC) contains all of the motifs that are characteristic of Cys loop channel subunits but cannot be assigned to any of the four established families on the basis of sequence similarity. Genes for ZAC are present in human and dog but appear to have been lost from mouse and rat genomes. Transcripts of ZAC subunits were detected in human placenta, trachea, spinal cord, stomach, and fetal brain. Transfection of human embryonic kidney cells with ZAC subunit cDNA caused expression of spontaneous current. By screening with a broad range of potential agonists and antagonists, we determined that tubocurarine inhibits the spontaneous current whereas Zn2+ activates the expressed receptors. The absence of Zn2+-activated channels in rats and mice may explain why this fifth member of the LGIC superfamily has evaded detection until now.