The cellular protein Cyclophilin A (Cyp A) is packaged into human immunodeficiency virus type 1 (HIV-1) particles through a specific interaction with the capsid domain of the Gag polyprotein. Inhibition of Cyp A incorporation by mutagenesis or cyclosporin treatment severely affects infectivity of all HIV-1 M subtypes tested. In contrast, the closely related lentiviruses HIV-2 and simian immunodeficiency virus (SIV) do not package Cyp A and are not inhibited by cyclosporin. For the HIV-1 group O isolate MVP5180, it was found that Cyp A incorporation and Cyp A dependence of infectivity did not correlate. This virus incorporates Cyp A but is not sensitive to treatment with cyclosporin A. For a more detailed study concerning the relationship between Cyp A incorporation and Cyp A dependence, we have analyzed five group O isolates for their ability to incorporate Cyp A and their sensitivity to cyclosporin treatment. All group O viruses incorporated Cyp A in comparable amounts as the M-group HIV-1 strain NL4-3. Furthermore, Cyp A incorporation was inhibited by cyclosporin in all cases. However, while isolate MVP 5180 was confirmed to replicate independent of Cyp A, three of the other four isolates were sensitive to cyclosporin treatment. Sequence analysis of the Cyp A binding regions revealed that the proline-rich motif, which is responsible for Cyp A incorporation, was conserved in all four isolates, while some sequence variations were detected in other positions close to this region. These results suggest that Cyp A dependence of replication is influenced by regions outside the Cyp-binding loop and may aid in determination of Cyp A function in HIV-1 replication.