Changes were determined in the activity of submandibular gland N-acetyl-beta-glucosaminidase from streptozotocin-induced diabetic and insulin-treated rats. Most of the activity of this enzyme was localized histochemically in the ductal cells. The activities of this enzyme from both the subcellular supernatant and lysosomal fractions were increased in the diabetic group, and recovered to the level of the controls in the insulin-treated group. Although sex differences were observed in the activity of this enzyme, with the activity in the male rats of the control group being lower than that in the females, these differences disappeared in the diabetic group, suggesting that insulin may be related to the expression of androgen function. Both enzymes in the supernatant and lysosomal fractions were separated by isoelectric focusing into two enzymatic proteins with isoelectric points in the vicinities of pI 3 and pI 8. The effects of diabetes were reflected in an increase primarily in the activity in the vicinity of the pI 8 isoenzyme in the supernatant fraction, and in an increase in both isoenzymes in the lysosomal fraction. It is clear from these findings that the diabetic condition brings about an insulin-dependent increase in the activity of N-acetyl-beta-glucosaminidase in the rat submandibular gland, and imparts certain changes in the properties of the enzymatic proteins themselves.