Recently, glycine has been shown to prevent liver injury after endotoxin treatment in vivo. We demonstrated that ethanol and endotoxin stimulated Kupffer cells to release prostaglandin E₂ (PGE₂) which elevated oxygen consumption in parenchymal cells. Since glycine has been reported to protect renal tubular cells, isolated hepatocytes and perfused livers against hypoxic injury, the purpose of this study was to determine if glycine prevents increases in intracellular free Ca²⁺ ([Ca²⁺]_i) in hepatocytes by agonists released during stress, such as with prostaglandin E₂ (PGE₂) and adrenergic hormones. Liver parenchymal cells isolated from female Sprague-Dawley rats were cultured for 4 hours in DMEM/F12 medium and [Ca²⁺]_i in individual cells was assessed fluorometrically using the fluorescent calcium indicator, fura-2. PGE₂ caused a dose-dependent increase in [Ca²⁺]_i from basal values of 130 ± 10 to maximal levels of 434 ± 55 nM. EGTA partially prevented this increase, indicating that either extracellular calcium or agonist binding is Ca²⁺ dependent. TMB-8, an agent that prevents the release of Ca²⁺ from intracellular stores, also partially blocked the increase in [Ca²⁺]_i caused by PGE₂, suggesting that intracellular Ca²⁺ pools are involved. Taken together, these results are consistent with the hypothesis that both the intracellular and extracellular Ca²⁺ pools are involved in the increase in [Ca²⁺]_i caused by PGE₂. Interestingly, glycine, which activates anion (i.e., chloride) channels, blocked the increase in [Ca²⁺]_i due to PGE₂ in a dose-dependent manner. Low-dose strychnine, an antagonist of glycine-gated chloride channel in the central nervous system, partially reversed the inhibition by glycine. When extracellular Cl^- was omitted, glycine was much less effective in preventing the increase in [Ca²⁺]_i due to PGE₂. Phenylephrine, an ₁-type adrenergic receptor agonist, also increased [Ca²⁺]_i as expected from 159 ± 20 to 432 ± 43 nM. Glycine also blocked the increase in [Ca²⁺]_i due to phenylephrine, and the effect was also reversed by low-dose strychnine. Taken together, these data indicate that glycine rapidly blocks the increase in [Ca²⁺]_i in hepatic parenchymal cells due to agonists released during stress, most likely by actions on a glycine-sensitive anion channel, and that this may be a major aspect of glycine-induced hepatoprotection.