FKBP25, a previously uncharacterized 25-kDa FK506- and rapamycin-binding protein, was purified to homogeneity from calf thymus, brain, and spleen, and the sequence of a 215 amino acid (aa) 24-kDa C-terminal peptide was established. The N-terminal domain (101 aa) is unrelated to any known protein, is hydrophilic, and is predicted by circular dichroism spectroscopy to be largely alpha-helix. The C-terminal domain (114 aa) is homologous to FKBP12 and other FKBPs but has a potential nuclear targeting sequence and a unique insertion of seven amino acids in one of its loops. FKBP25 displays the rotamase activity characteristic of FKBPs; the activity is inhibited by the immunosuppressants rapamycin (Ki = 0.9 nM) and FK506 (Ki = 160 nM), but not cyclosporin A. The protein, its rapamycin selectivity, and the potential nuclear targeting sequence are discussed in terms of the structure of hFKBP12.