The ability of two radiolabeled, photoreactive estrogen analogues, [3H]hexestrol diazoketopropyl ether ([3H]Hex-DKP) and [3H]hexestrol azide ([3H]Hex-N3), to covalently label the uterine estrogen receptor is studied. Lamb uterine estrogen receptor preparations that have been partially purified (ammonium sulfate precipitation, Sephadex G-200 chromatography) and disaggregated by limited trypsinization can be electrophoresed on polyacrylamide gels under conditions where binding activity is preserved. This electrophoretic procedure was used to fractionate the proteins labeled by the two photoreactive estrogen analogues. Prior to photolysis, peaks of radioactivity indicating estrogen specific binding of [3H]-Hex-N3 and [3H]Hex-DKP are evident on the gels, although dissociation of the latter compound is extensive. When preparations of uterine estrogen receptor that contain the photoreactive derivatives are irradiated and then electrophoresed, reversibly labeled proteins can be distinguished from irreversibly labeled ones (covalently bonded), by extraction of the individual gel slices with organic solvents. While no irreversible binding to receptor appears to result from irradiation with [3H]Hex-DKP, irradiation with [3H]Hex-N3 does covalently label the estrogen receptor. The receptor covalently labeled with [3H]Hex-N3 has the same electrophoretic mobility as the unlabeled receptor; the covalent labeling process is estrogen-site specific, and the efficiency of labeling (15-20%) is consistent with the inactivation efficiency of Hex-N3, previously measured by an indirect assay. This is the first example of the labeling of a steroid hormone receptor by photoaffinity labeling.