The affinity labels 11 alpha- and 16 alpha-(bromo[2'-3H]acetoxy)progesterone (BAP) react covalently with amino acids present in the progesterone binding site of the human uterine progesterone receptor. Hydrolysis of the affinity labeled receptor followed by separation and analysis of the amino acid products demonstrated the sites of affinity labeling. The 11 alpha-BAP alkylates the 1-position of a histidine residue. The 16 alpha-BAP alkylates the 3-position of histidine, and a methionine residue. Affinity labeling did not occur in the presence of excess progesterone, and under the optimum conditions for affinity labeling of the receptor, heat-denatured receptor, bovine serum albumin, and 20 beta-hydroxysteroid dehydrogenase were not affinity labeled. This is the first report of the identification of specific amino acid residues in the binding site of a steroid hormone receptor.