Histidine residues in immunophilins, particularly His-126 of cyclophilin (CyP) and His-87 of the FK506 binding protein (FKBP), have been suggested to play important roles in ligand binding and peptidyl prolyl cis-trans isomerase (PPiase) catalysis. The charged states of the histidine residues in FKBP and CyP, which were characterized by their pKa values, have been determined in the absence and presence of the immunosuppressant ligands, ascomycin and cyclosporin A (CsA), respectively, by using a heteronuclear two-dimensional NMR method. Overall, the histidine residues in FKBP and CyP are very acidic with pKa values ranging from < or = 2.8 to 6.5, indicating that they are predominantly uncharged at physiological pH. To our knowledge, the pKa value of < or = 2.8 determined from this study is the lowest pKa reported for the free imidazole ring of the histidine residues in proteins. The abnormally acidic pKa's of His-25 in FKBP and His-54 in CyP could be explained by their highly positively charged environments. His-87 of FKBP, which is located in the FK506 binding pocket, was found to exist in two forms in free FKBP with pKa values of 5.9 and 6.5 for the major and minor forms, respectively. His-126, which is part of the CsA and substrate binding site, has a pKa of 6.3 in free CyP. The pKa values of these two histidine residues in the free proteins are higher than the pKa's obtained for the peptidyl prolyl cis-trans isomerase (PPiase) activity of these enzymes, indicating that the acid/base characters of His-87 of FKBP and His-126 of CyP are not essential in the PPiase catalysis. The hydrogen bonding of the histidine imidazole rings and the effect of hydrophobicity upon changes in pKa values are discussed.