Histamine has been shown to play a role in arthropod vision; it is the major neurotransmitter of arthropod photoreceptors. Histamine-gated chloride channels have been identified in insect optic lobes. We report the first isolation of cDNA clones encoding histamine-gated chloride channel subunits from the fruit fly Drosophila melanogaster. The encoded proteins, HisCl1 and HisCl2, share 60% amino acid identity with each other. The closest structural homologue is the human glycine ␣3 receptor, which shares 45 and 43% amino acid identity respectively. Northern hybridization analysis suggested that hisCl1 and hisCl2 mRNAs are predominantly expressed in the insect eye. Oocytes injected with in vitro transcribed RNA, encoding either HisCl1 or HisCl2, produced substantial chloride currents in response to histamine but not in response to GABA, glycine, and glutamate. The histamine sensitivity was similar to that observed in insect laminar neurons. Histamine-activated currents were not blocked by picrotoxinin, fipronil, strychnine, or the H2 antagonist cimetidine. Co-injection of both hisCl1 and hisCl2 RNAs resulted in expression of a histamine-gated chloride channel with increased sensitivity to histamine, demonstrating coassembly of the subunits. The insecticide ivermectin reversibly activated homomeric HisCl1 channels and, more potently, HisCl1 and HisCl2 heteromeric channels.Histamine has been recognized for several years as the major neurotransmitter of arthropod photoreceptors (1, 2). In Drosophila, immunocytochemistry has shown high levels of histamine in photoreceptors and their synapses (2, 3). Further, histidine decarboxylase activity has been demonstrated in Drosophila photoreceptors, suggesting that histamine is synthesized in these cells (2). Drosophila mutants that are deficient in the hdc gene coding for histamine decarboxylase have been identified (4), and flies homozygous for the null mutation appear to be blind (5). Although light-dependent release of histamine from photoreceptors has not yet been demonstrated, exposure of the fly postsynaptic neuron to histamine mimics the effects of light (6).Phototransduction of invertebrates has been most extensively studied in insects, but histamine has been implicated as a neurotransmitter in photoreceptors of several other invertebrate species including barnacles and the horseshoe crab Limulus (7,8), whereas glycine and GABA 1 function as the inhibitory neurotransmitters in vertebrate phototransduction (9). Outside of phototransduction, histamine also appears to be the neurotransmitter in some mechanosensory neurons in Drosophila (10) and in lobster stomatogastric, cardiac, and olfactory neurons (11-13). To date, the molecular nature of the histamine receptor in these neurons is unknown. The laminar neurons that are postsynaptic to insect photoreceptors respond to light with a rapid, chloride-mediated hyperpolarization that can be mimicked by application of histamine (6). These laminar neurons were isolated from optic lobes of several insect species, and the hista...