Photooxidation of estradiol receptor form MTW9 rat mammary tumor cytosol leads to a time-dependent loss of DNA-binding activity; this effect is pH-dependent. Binding of receptor to DNA does not protect the DNA-binding site from photooxidation. The estrogen binding site of the receptor is also susceptible to photooxidation, although the rate of loss of estradiol binding capacity is considerably less than that of DNA-binding capacity; this process is independent of pH. Binding of receptor to estradiol substantially protects the estradiol binding site from photooxidation. Sucrose gradient analysis of photooxidized receptor preparations showed no qualitative difference compared with nonirradiated controls. Similarly, the isoelectric focusing pattern of photooxidized receptor showed two peaks of radioactivity at pH approximately 5.6 and 6.3, as did nonirradiated receptor.