The hormonal regulation of trypsin-like esteroprotease synthesis in mouse submandibular gland was studied at the isozyme level. Antiserum to a mixture of two purified esteroproteases precipitated all the esteroproteases in a crude extract of this gland. Measurement of incorporation of [3H]leucine showed that total esteroprotease synthesis was stimulated by both 5 alpha-dihydrotestosterone and triiodothyronine and that the two hormones had synergistic effects. The observed correlation between the increases of synthetic rate and specific activity of this enzyme suggests that the enzyme level is regulated mainly by the rate of enzyme synthesis. Newly synthesized esteroprotease-antibody complexes gave four peaks of radioactivity with esteroprotease activity and one peak without enzyme activity on isoelectric focusing in acrylamide gel containing 8 M urea. The radioactivities of these five peaks were increased similarly by the two hormones separately or in a combination. These results suggest that the actions of androgens and thyroid homrones in esteroprotease synthesis are indistinguishable at the isozyme level.