Isolated olfactory receptor neurons from the squid Lolliguncula brevis respond to betaine, a repellent odorant, with hyperpolarizing receptor potentials. Using perforated-patch techniques, we determined that the hyperpolarizing conductance was selective for Cl- and could be reversibly blocked by the Cl- channel blockers 4-acetamido-4'-isothio-cyanatistilbene-2,2'disulfonic acid and niflumic acid. Gramicidin-patch recordings revealed that [Cl-]i in squid olfactory receptor neurons is normally very low compared to vertebrate olfactory receptor neurons, and that activating a Cl- conductance would hyperpolarize the cell in vivo. The lack of dependence on internal or external K+ or Na+ ruled out the possibility that the Cl- conductance was generated by a cation-dependent cotransporter or pump. Common G-protein-dependent signalling pathways, including phospholipase C, arachidonic acid, and cyclic nucleotides, do not appear to be involved. Ca2+ imaging experiments showed that betaine did not affect [Ca2+]i, suggesting that the Cl- current is not Ca2+ dependent. Our findings represent the first report of an odorant-activated, hyperpolarizing chloride conductance in olfactory receptor neurons.