The mechanisms by which cyclophilin A (CypA) governs hepatitis C virus (HCV) replication remain unknown. Since CypA binds two essential components of the HCV replication complex (RC)--the polymerase NS5B and the phosphoprotein NS5A--we asked in this study whether CypA regulates their RC association. We found that CypA, via its isomerase pocket, locates in a protease-resistant compartment similar to that where HCV replicates. CypA association with this compartment is not mediated by HCV. Moreover, CypA depletion of RC does not influence NS5A and NS5B RC association, arguing against a model where CypA governs HCV replication by recruiting NS5A or NS5B into RC.