FKBP-12, the major T-cell binding protein for the immunosuppressive agents FK506 and rapamycin, catalyzes the interconversion of the cis and trans rotamers of the peptidyl-prolyl amide bond of peptide and protein substrates. The function of rotamase activity in cells and the role of FKBP-12 in immunoregulation is uncertain. In this paper we report the cloning and characterization of the human chromosomal FKBP-12 gene and four processed FKBP-12 pseudogenes. The FKBP-12 gene is 24 kilobases in length and contains five exons. The protein-coding region of the gene is divided into four exon modules that correlate with the structural and functional domains of the protein. The novel structure of FKBP-12 resulting from the topology of the antiparallel beta-sheet is the topological crossing of two loops that are encoded by separate exons. Separate exons also encode the antiparallel beta-sheet and alpha-helical region that define the drug-binding pocket and enzyme activity site of FKBP-12. The exon organization of the FKBP-12 gene also provided insight into the genetic evolution of the immunophilin family. Knowledge of the FKBP-12 gene structure will enable inactivation of this gene by homologous recombination in cells to provide a model to study the role of FKBP-12 in immunoregulation and normal cellular processes.