Microbial milk-clotting enzymes are valued as calf rennet substitutes in the cheese industry. Mucor pusillus QM 436 was identified to produce the highest milk-clotting activity during screening of 19 fungal strains. Salted whey resulting from Ras (Cephalotyre) cheese manufacture as a whole medium for growth of Mucor pusillus QM 436 and production of the enzyme. The milk-clotting enzyme from Mucor pusillus QM 436 was purified to 7.14-fold with 54.4% recovery by precipitation in ammonium sulfate, ethanol and fractionated by gel filtration on Sephadex G-100. The enzyme was active in the pH range 5.5-7.5 and was inactivated completely by heating 5 min at 70ºC and 30 min at 65ºC.  The highest level of enzyme activity was obtained at 60ºC, pH 5.5. A positive and proportional relationship occurred in presence of CaCL2 in milk, with inhibition occurred in presence of NaCl. The high level of milk-clotting activity coupled with a low level of thermal stability suggested that the milk-clotting enzyme from Mucor pusillus QM 436 should be considered as a potential substitute for calf rennet.