Fungal milk-clotting enzymes have gained value as bovine Chymosin substitutes in the cheese industry. In this work, the effects of culture conditions on the production of extracellular milk clotting enzymes from Mucor mucedo DSM 809 in submerged fermentation were studied. The maximum activity was observed after 48 h of cultivation at 24 degrees C in Erlenmeyer flasks. The optimized initial pH and shaking speed for enzyme production were 4.5 and 220 rpm, respectively. Glucose at a concentration of 1% (w/ v) was the best carbon source for the production of enzyme among the carbohydrates examined (glucose, fructose, lactose, maltodextrin). On the other hand casein at a concentration of 0.5% (w/v) was the selected nitrogen source in the media formulation. Under optimized conditions enzyme levels reached 130 SU per ml fermentation broth. The inoculum type and size has also affected biomass production and the biosynthesis of the enzyme. The preferred method was the inoculation of the culture media with spores at a total load of 6x10(5) spores per flask.