Glycine, strychnine and certain chloride channel blockers were reported to protect cells against lethal cell injury. These effects have been attributed to interactions with membrane proteins related to CNS glycine gated chloride channel receptors. We have investigated the pharmacology of these actions. Madin-Darby canine kidney (MDCK) epithelial cells were depleted of adenosine triphosphate (ATP) by incubation in glucose free medium containing a mitochondrial uncoupler. Medium Ca2+ was adjusted to 100 nM in the presence of an ionophore such that intracellular Ca2+ did not increase, and Ca(2+)-related injury mechanisms were inhibited. This permitted more sensitive quantitation of protection against cell injury attributable to glycine or other agents whose actions might be related to those of the amino acid. Two classes of compounds showed cytoprotective activity in this system: (1) ligands at chloride channel receptors, such as glycine, strychnine and avermectin B1a; (2) chloride channel blockers, including cyanotriphenylboron and niflumic acid, both of which are known to bind to channel domains of CNS glycine receptors. Morphological and functional studies showed that the compounds preserved plasma membrane integrity, but permitted cell swelling. Substitution of medium chloride by gluconate, or chloride salts by sucrose, did not substantially modify lethal damage or its prevention by glycine or other drugs. The compounds did not modify ATP declines. At least for some compounds, cytoprotection appeared to be specific to structural features on the molecules. These observations are consistent with the hypothesis that a plasma membrane protein related to glycine-gated chloride channel receptors plays a significant role in cell injury, but indicate that the mechanisms of injury and protection by compounds active in this system are not related to chloride fluxes.