A non-toxic potent anticoagulant metalloprotease NN-PF3 has been purified to homogeneity from the Indian cobra (Naja naja naja) venom through a combination of column chromatography and electrophoresis. NN-PF3 is a single chain protein with a molecular weight of 68 kDa by SDS–PAGE. It hydrolysed casein, gelatin, haemoglobin and bovine fibrinogen, but did not hydrolyse bovine serum albumin or synthetic substrates such as TAME, BAEE and BAPNA. EDTA, EGTA and cyanide inhibited the enzymatic activity while 1,10-phenanthroline, PMSF, leupetin and pepstatin did not show any effect. NN-PF3 is a metalloprotease containing Ca2+ and Zn2+ at a molar ratio of 1:1.2 and 1:0.4, respectively, as revealed by atomic absorption spectroscopy. NN-PF3 was non-lethal up to an i.p. dose of 15 mg/kg body weight of mice and is devoid of myotoxicity, cytotoxicity and haemorrhagic activity. It is weakly oedematic, but strongly anticoagulant in property and the effect observed was both dose and time dependent.