The cytosolic concentration, Mr, and isoforms of cyclophilin (CyP), a specific cytosolic binding protein for cyclosporin A (CsA), were determined in normal and neoplastic human tissues as well as tissues from species of diverse phylogeny. CyP was present in all tissues examined; however, concentrations varied significantly among different tissue types. The CyP concentration was highest in lymphoblasts from a patient with T cell acute lymphocytic leukemia (1.15 micrograms/mg protein) and Hodgkin's and non-Hodgkin's lymphomas. CyP concentration in colon adenocarcinomas was twofold to threefold greater than that found in adjacent normal tissue. CyP from all normal and neoplastic human tissues examined had an apparent Mr of 17,000 determined by gel filtration HPLC. Major (pI 8.6 to 8.7) and minor (pI 6.7 to 6.9) CyP isoforms were identified in all human and murine tissue extracts by column sucrose gradient isoelectrofocusing; however, the ratio of the major to minor isoform varied widely. Among other species examined, significant concentrations of CyP were detected in cytosol extracts from sponges (Microciona prolifera), yeast (Saccharomyces cerevisiae), mushrooms, the giant cockroach (Blaberus discoidalis), and a trematode (Schistosoma mansoni). By contrast, CyP was not detectable in extracts of Escherichia coli. A twofold to threefold elevation in the CyP content of murine splenocytes was detected 72 hr after Con A stimulation. A survey of a variety of natural products, synthetic compounds, and immunoregulating agents has failed thus far to identify compounds capable of competing with CsA for binding to CyP. The broad tissue and phylogenetic distribution of CyP, its highly conserved structure, and its increased content after mitogenic stimulation suggest a fundamental role in cellular metabolism.