The development of trypsin-like esteroproteases in the submandibular gland of mice was studied, using a newly synthesized naphthyl ester (tosyl-L-lysine alpha-naphthyl ester) for the preparation of zymograms and for histochemical demonstration of the enzyme. Esteroprotease activities were first detected spectrophotometrically on day 15 after birth; then increased markedly after day 20. A sex difference in esteroprotease activity appeared on day 25. Zymograms prepared after isoelectric focusing in polyacrylamide slab gels showed that the glands of neonatal mice contained esteroproteases with a rather different composition from that of adult mice. The adult type isozymes appeared first on day 15, and their activities increased markedly after day 20. Histochemical studies revealed that the isozymes of neonatal mice were derived from mast cells. A few striated ducts were first stained on day 15, and the sex difference of the granular tubules became obvious on day 25. These results indicate that the development of trypsin-like esteroproteases faithfully reflects the differentiation of granular tubules in the mouse submandibular gland, except in the neonatal period.