A sensitive method for quantitative determination of esterase activity has been applied to human saliva and used for substrate and inhibitor studies. By means of gel isoelectrofocusing the whole saliva esterases were divided into two main groups, each resolved into several bands. Comparison of isoelectrophoretic zymograms of whole, submandibular and parotid saliva indicates that one of the groups originates from the submandibular fluid. With the aid of gel filtration and ion-exchange chromatography the saliva esterases were partially separated and purified. The classification of the main saliva esterases as carboxylesterases (EC 3.1.1.1) is discussed in terms of their substrate and inhibitor properties.