The cDNA encoding a human cytosolic 40-kDa cyclophilin (CyP-40) has been inserted into a modified pGEX-3X expression vector and expressed in Escherichia coli to yield recombinant CyP-40 at levels up to 4 mg/l medium. The protein was purified to homogeneity using a cyclosporin affinity matrix and gel filtration. The recombinant CyP-40 showed peptidyl-prolyl cis-trans isomerase activity (kcat/Km = 1.12 x 10(6) M-1.s-1) comparable to that of bovine brain CyP-40. The weak affinity of CyP-40 for cyclosporin A was postulated to arise from a histidine residue that replaces a tryptophan residue critical for cyclosporin A binding and highly conserved in other cyclophilins that have high affinity for cyclosporin A. Site-directed mutagenesis to replace His141 by tryptophan yielded a protein with an approximately 20-fold greater affinity for cyclosporin A (Kdapp 11.5 +/- 2 nM as determined by tryptophan fluorescence measurements). The intrinsic isomerase activity of this mutant protein with succinyl-Ala-Ala-Pro-Phe 4-nitroanilide as substrate was about nine times greater than the value obtained for the nonmutated recombinant CyP-40 and had an activity similar to that of CyP-18. NMR difference spectroscopy and molecular modelling revealed a cyclosporin-A-binding domain that is similar to that of CyP-18.