The coagulant retained in cheese curd is a major contributor to proteolysis during ripening. The objective of this study was to quantify the effects of several milk-related factors and parameters during cheese manufacture on the retention of coagulant in cheese curd. The amount of coagulant retained in curd was determined by its activity on a synthetic heptapeptide (Pro-Thr-Glu-Phe-[NO2-Phe]-Arg-Leu) using reversed-phase HPLC. The retention of chymosin in cheese curd increased significantly when the pH of milk was reduced at rennet addition below pH 6.1, the pH at whey drainage below pH 5.7, or the average casein micelle size in milk and when the ionic strength of milk was increased. The casein content of milk and the quantity of chymosin added to milk had no significant effect on the retention of chymosin in curd; the quantity of coagulant bound per gram of casein remained unchanged.