A partially purified trypsin-like protease was isolated from the human uterine cytosol that transformed the rat uterine estrogen receptor-[3H]estradiol complex from an 8S to a 4.5S sedimenting estrogen-binding protein, on sucrose gradient analysis. The transformation of the rat uterine estrogen receptor took place preferentially when the [3H]estradiol was associated with the estrogen receptor, rather than in the unassociated state. Alteration of the molecular size occurred without loss of the [3H]estradiol-binding capacity of the estrogen receptor, indicating that a limited proteolysis of the estrogen receptor-[3H]estradiol complex was occurring. The human uterine protease was inhibited by diisopropylfluorophosphate and tosyl-lysine chloromethyl ketone, and hydrolyzed benzoyl-arginine nitroanilide, indicating a trypsin-like activity. These data indicate the existence of a human uterine protease capable of altering the sedimentation form of the estrogen receptor-[3H]estradiol complex with high specificity. (Endocrinology93: 210, 1973)