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 PGE₂, which elevated oxygen consumption in parenchymal cells. Because 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 whether glycine prevents increases in intracellular free Ca²⁺ concentration ([Ca²⁺]_i) in hepatic parenchymal cells by agonists released during stress, such as with PGE₂ and adrenergic hormones. Liver parenchymal cells isolated from female Sprague-Dawley rats were cultured for 4 h 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. 8-(Diethylamino)octyl 3,4,5-trimethoxybenzoate (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. 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. 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.