Substrate specificities, as reflected in kc/Km, were determined for the peptidyl prolyl cis-trans isomerase activities of cyclophilin and the FK-506 binding protein (FKBP). The substrates investigated were peptides of the general structure Suc-Ala-Xaa-Pro-Phe-p-nitroanilide, where Xaa = Gly, Ala, Val, Leu, Phe, His, Lys, on Glu. While kc/Km for cyclophilin-catalyzed isomerization shows little dependence on Xaa, kc/Km values for FKBP-catalyzed isomerization display a marked dependence on Xaa and vary over 3 orders of magnitude. An important outcome of this work is the discovery that Suc-Ala-Leu-Pro-Phe-pNA is a reactive substrate for FKBP (kc/Km = 640,000 M-1 s-1). This substrate can be used with FKBP concentrations that are low enough to allow, for the first time, accurate determinations of Ki values for tight-binding inhibitors of FKBP. Using this new assay, we found that FK-506 inhibits FKBP with Ki = 1.7 +/- 0.6 nM. The results of this work support the hypothesis that cyclophilin and FKBP are members of a family of peptidyl prolyl cis-trans isomerases and that the members of this family possess distinct substrate specificities that allow them to play diverse physiologic roles.