An arginyl esterase, identified by hydrolysis of tosyl-L-arginine-O-methyl ester (TAME), is present in estrogen-stimulated, immature rat uterine cytosol. The esterase is Ca2+-dependent and appears 3-8 h after injection of 4.5 micrograms of 17 beta-estradiol (E2). The Ca2+-independent TAME hydrolase activity in stimulated uteri decreases as the Ca2+-dependent hydrolase activity increases. The TAME hydrolase activity is not increased by dexamethasone or testosterone, nor is it elevated in liver cytosol of the same E2-treated rats. Rat plasma contains a potent Ca2+-dependent TAME hydrolase activity distinguished from the uterine enzyme by its response to high Ca2+ concentrations and its interaction with goat anti-rat serum immunoglobulin G. The uterine TAME hydrolase is apparently a trypsin-like serine protease, as defined by its pattern of inhibition, e.g. response to diisopropylfluorophosphate. The Ca2+-dependent hydrolase kinetics are complex, with an initial lag phase followed by a dramatic acceleration in the rate of hydrolysis. Both the length of the lag phase and the extent of acceleration vary with the amount of cytosol. The metal requirement is specific for Ca2+. The endogenous substrate of this newly recognized, estrogen-stimulated TAME hydrolase and its role in uterine development remain to be elucidated.