Cyanide is a mitochondrial poison and its toxicity is mediated through histotoxic hypoxia. Although cyanide is regarded as a neurotoxin, its other toxic manifestations are also well documented. Cyanide triggers all those events which can lead to DNA damage, but its genotoxic potential has not been established yet. The present investigation addresses the DNA damage induced by cyanide in rat thymocytes in vitro. Cell viability (eosin Y exclusion and LDH leakage) along with DNA strand breaks were measured in thymocytes exposed to 1.25-10 mM KCN for various time intervals. Cleavage into oligonucleosomal fragments of extracted DNA from cyanide treated thymocytes were visualized on gel electrophoresis. Cyanide produced both time and dose dependent DNA fragmentation accompanied by cytotoxicity. The DNA damage was sensitive to elevated levels of extracellular Ca2+ and was minimal in Ca2+ free medium. The DNA fragmentation was attenuated by Zn2+ (modulator of Ca2+/Mg2+-dependent endonuclease), N-acetylcysteine (free radical scavenger) and diltiazem (Ca2+ channel blocker). Cyanide induced DNA damage was further observed in baby hamster kidney cells (BHK-21), where unlike thymocytes, internucleosomal DNA fragmentation was not observed. Thymocytes were more sensitive to cyanide as compared to BHK-21 cells.