Proteolytic enzymes, protease and trypsin have recently been introduced to reduce the inconsistency hitherto encountered in the unlabelled antibody--enzyme method using PAP. This study investigated factors determining the optimum conditions for use of such enzymes in order to establish which one is most suitable. Trypsin was the most effective enzyme; however, its activity decreased over 3 h, a feature paralleled immunocytochemically. Method and duration of fixation appears to influence the required time of exposure to trypsin in order that consistent immunostaining may be produced. Treatment of sections with trypsin prior to the use of the unlabelled antibody--enzyme method using PAP renders the technique reliable, provided the enzyme is used in a carefully controlled manner.