Batch shake flask experiments were carried out with a fungal strain of Mucor miehei ATCC 3420 to study the effects of five fermentation parameters; initial D-glucose concentration, temperature, initial pH, agitation rate and inoculum ratio, on the production of rennin in a chemically defined medium. A statistical design was used to assign the experimental levels of five fermentation parameters for the determination of optimum conditions. The highest concentration of midi-clotting activity (MCA) obtained was 2286 SU cm(-3) (without concentration of the broth) at 29.6 g dm(-3) initial D-glucose concentration, 6.8 initial pH, 37.6 degreesC temperature, 81 stroke per min agitation rate and 5.2% (v/v) inoculum ratio. Although enzyme secretion was a function of all the operational parameters investigated, the pH range (4.6-5.2) reached at the time of enzyme production had a profound effect on milk-clotting activity levels. In the fermentation sample at maximum milli-clotting activity the R factor, the ratio of milk-clotting activity to proteolytic activity, was determined as 1 SU PU-1, denoting similar characteristics to a commercial animal rennet.