The activity of bile-salt-stimulated esterase was monitored in human milk during storage at room temperature, in a refrigerator (4 degrees C), in a freezer (-19 degrees C), and in milk that had been freeze dried and rehydrated after storage of the lypophilized milk at 4 degrees C and -19 degrees C. In all cases, there was some loss of activity, independent of measurement in the presence or absence of sodium taurocholate, and the loss was highest in milk stored at room temperature, approximating a 50% loss in 1-2 days. Activation and inactivation of solutions of the purified enzyme have been identified by measuring its esterase activity against a variety of 4-nitrophenylalkanoate esters at pH 7.3 within the temperature range 5-46.5 degrees C. The optimum temperature was 42 degrees C. The presence of bile salt partially serves to maintain the conformation of the bile-salt-binding site as the esterolytic binding site becomes inactivated at 50 degrees C. Bile salts thus exert a positive template effect on the enzyme.