The 1,25-dihydroxyvitamin D3 receptor is a member of the steroid/thyroid hormone receptor gene family and is thought to act by regulating transcription of specific genes. In this report, we have used peptide mapping of porcine 1,25-dihydroxyvitamin D3 receptor to localize the sites of phosphorylation, photoaffinity labeling, and monoclonal antibody binding. Receptor was immunoprecipitated from [32P]orthophosphate-labeled pig kidney LLC-PK1 cells grown in the absence and presence of 1,25-dihydroxyvitamin D3. Phosphorylation of receptor was induced by 1,25-dihydroxyvitamin D3. The phosphorylated receptor was digested with Staph A V8 protease within Cleveland gels and the 32P label was found entirely in a 23-kDa fragment. Similarly, receptor that was photoaffinity labeled with 1,25-dihydroxy-[26,27-3H]vitamin D3 was subjected to peptide mapping by Cleveland gels. The primary site of photoaffinity label incorporation was in the same 23-kDa peptide. This peptide was localized to a region in the center of the receptor protein, spanning part of the previously designated hinge region and roughly one-half the proposed steroid binding domain. Because phosphorylation did not occur in the DNA binding domain, it may not be involved in the binding of receptor to DNA. The localization of phosphorylation sites to this 23-kDa peptide may suggest that phosphorylation is involved in steroid binding and/or activation of the receptor.