The nucleomyofibrillar fraction of epididymides from sexually mature rabbits contains a novel leupeptin-sensitive protease that disrupts the oligomeric conformation of cytosolic estrogen and progesterone receptors, and molybdate inhibits this process. In this report we used the AtT-20 cell glucocorticoid receptor as substrate and performed analyses under nondenaturing vs. denaturing conditions to further investigate the effects of the epididymal protease and molybdate on steroid receptor structure. Analysis on low salt sucrose gradients indicated that the protease partially converted the oligomeric (9-10S) glucocorticoid receptor to several more slowly sedimenting forms (3-7S), and this effect was not observed in the presence of molybdate. Paradoxically, gradient analysis under high salt conditions revealed that the protease induced a discrete, quantitative and molybdate-insensitive conversion of the 4-5S steroid-binding subunit to a 3S form. Further studies were done using denaturing polyacrylamide gel electrophoretic analysis of receptor that had been labeled covalently with [3H]dexamethasone 21-mesylate and partially purified by DNA/cellulose chromatography. At 0-4 C, the protease cleaved the steroid-binding subunit (mol wt, 96,900) of the receptor to a single steroid-labeled fragment (mol wt, 42,600). Under these conditions, digestion was complete within 30-60 min and was inhibited by leupeptin, but was unaffected by thiol-reactive reagents or molybdate. The epididymal protease and alpha-chymotrypsin produced steroid-labeled receptor fragments that were indistinguishable in size, shared an epitope recognized by our BuGR-2 monoclonal antibody, and retained DNA-binding activity. Despite the apparent similarity of these two enzymes, they are distinct, since the chymotrypsin-dependent cleavage event was not inhibited by leupeptin. These studies show that the epididymal protease attacks a site on the steroid-binding subunit of glucocorticoid receptors as well as estrogen and progestin receptors. It also appears that the cleavage site is situated close to that most readily attacked by alpha-chymotrypsin. Finally, our data provide independent confirmation of a recent report indicating that molybdate ions interact directly with the cytosolic steroid receptor to stabilize its oligomeric structure even after proteolysis within the steroid-binding subunit.