Cyclophilins are folding helper enzymes belonging to the class of peptidyl-prolyl cis-trans isomerases (PPIases; EC 5.2.1.8) that catalyze the cis-trans isomerization of peptidyl-prolyl bonds in proteins. They are ubiquitous proteins present in almost all living organisms analyzed to date, with extremely rare exceptions. Few cyclophilins have been described in Actinobacteria, except for three reported in the genus Streptomyces and another one in Mycobacterium tuberculosis. In this study, we performed a complete phylogenetic analysis of all Actinobacteria cyclophilins available in sequence databases and new Streptomyces cyclophilin genes sequenced in our laboratory. Phylogenetic analyses of cyclophilins recovered six highly supported groups of paralogy. Streptomyces appears as the bacteria having the highest cyclophilin diversity, harboring proteins from four groups. The first group was named "A" and is made up of highly conserved cytosolic proteins of approximately 18 kDa present in all Actinobacteria. The second group, "B," includes cytosolic proteins widely distributed throughout the genus Streptomyces and closely related to eukaryotic cyclophilins. The third group, "M" cyclophilins, consists of high molecular mass cyclophilins ( approximately 30 kDa) that contain putative membrane binding domains and would constitute the only membrane cyclophilins described to date in bacteria. The fourth group, named "C" cyclophilins, is made up of proteins of approximately 18 kDa that are orthologous to Gram-negative proteobacteria cyclophilins. Ancestral character reconstruction under parsimony was used to identify shared-derived (and likely functionally important) amino acid residues of each paralogue. Southern and Western blot experiments were performed to determine the taxonomic distribution of the different cyclophilins in Actinobacteria.