The effects of ionizing irradiation on the sedimentation coefficients of both estrogen receptor (ER) and progesterone receptor (PgR) have been examined in comparison to the effects of proteolysis. DMBA-induced rat mammary tumors were subjected to a treatment of 20 Gy and the ER and PgR concentrations were determined at different time intervals after irradiation. On a 5-20% sucrose gradient the ER sedimented as 9-11 and 4-5 S molecular forms, while PgR sedimented as a small 8-9 S peak and a major 4-5 S peak. Radiotherapy particularly reduced the 4-5 S sedimentation peaks of both receptors but did not initiate any new sedimentation forms. Although the 4-5 S ER receptor concentrations remained low, both progesterone receptor forms appeared to recover by 60 days after treatment. As these effects could be due to the release of proteolytic enzymes following irradiation of tumors, the receptors from untreated tumors were exposed to different concentrations of trypsin. The effects of trypsin were identical for ER and for PgR, and proved to be dependent on the trypsin concentration. Only concentrations of trypsin up to 30 micrograms/ml resulted in a reduction of 9-11 S ER or 8-9 S PgR forms which was accompanied by a simultaneous increase in the 4-5 S peaks, resulting in no change in total binding sites. Still higher trypsinization (300-3000 micrograms/ml) also reduced the 4-5 S ER and PgR fractions. In the presence or the absence of sodium molybdate, a stabilizer of the faster sedimenting forms of the receptor, no alterations were observed in the position of, or the total number of binding sites of, the sucrose gradient fractions from control or irradiated tumors. The irradiation effects appear to be due either to damage of the cytosolic ER receptor, thereby preventing its participation in the induction of de novo synthesis of ER and PgR, or to the non-specific damage of transcription and/or translation systems.