The relationship between the actions of androgen and thyroid hormone in induction of trypsin-like esteroproteases in mouse submandibular gland was studied. Zymograms prepared using tosyl-L-lysine alpha-naphthyl ester as substrate after isoelectric focusing in polyacrylamide slab gel showed that the isozymes of trypsin-like esteroprotease induced by 5 alpha-dihydrotestosterone and triiodothyronine were identical. The time courses of esteroprotease induction by these hormones were very similar, and the lag time was not shortened by treatment with both hormones. The doses of 5 alpha-dihydrotestosterone and triiodothyronine for half-maximal induction were 0.5 mg and 2.5 micrograms per 100 g body weight, respectively, and these values were not altered by simultaneous injection of other hormones. The binding capacity and affinity of androgen receptor for methyltrienolone, a synthetic androgen, were not affected by daily injections of triiodothyronine for 5 days. These results suggest that androgen and thyroid hormone act independently, not competitively, and so the two hormones induce trypsin-like esteroproteases additively.