Cyanide is primarily a neurotoxin but its hepatotoxic and nephrotoxic potentials are also known. The present study reports the effect of alpha-ketoglutarate A-KG (2.5-20 mM; 0 min), a potential cyanide antidote on potassium cyanide (KCN; 1.25-20 mM) induced cytotoxicity in primary culture of rat hepatocytes. Cytotoxicity measured at various time points (0.5-24 h), was characterized by increased leakage of intracellular lactate dehydrogenase, alanine aminotransferase and aspartate aminotransferase, accompanied by diminished mitochondrial function (MTT assay), mitochondrial membrane potential (Rhodamine 123 assay), and ATP levels. However, lipid peroxidation (malondialdehyde assay) and DNA damage were not observed. In a separate study, levels of cyanide, A-KG and thiocyanate were measured in the culture medium of hepatocytes, treated with KCN (5 mM) and/or A-KG (5 or 10 mM; 0 min), and in the serum of rats given oral treatment of KCN (10 mg/kg) and/or A-KG(0.5, 1 or 2 g/kg; 0 min). Cyanide and A-KG interaction was best exhibited when both were added in equimolar dose in vitro. In rats, cyanide levels were significantly reduced by 1 and 2 g/kg A-KG. It can be concluded from the results that, (i) a very high dose of cyanide is required to produce cytotoxicity and other cellular perturbations in rat hepatocytes, (ii) cytotoxicity is independent of lipid peroxidation and DNA damage, (iii) A-KG provides significant protection against cyanide, particularly at equimolar dose in vitro, and (iv) a very high dose of A-KG is required for cyanide detoxification in vivo, suggesting that the dose of A-KG could be reduced by improving its bio-availability.