The human immunodeficiency virus type 1 (HIV-1) Gag polyprotein binds to cyclophilin A and incorporates this cellular peptidyl prolyl-isomerase into virions. Disruption of cyclophilin A incorporation, either by gag mutations or by cyclosporine A, inhibits virion infectivity, indicating that cyclophilin A plays an essential role in the HIV-1 life cycle. Using assays for packaging of cyclophilin A into virions and for viral replication sensitivity to cyclosporine A, as well as information gleaned from the alignment of Gag residues encoded by representative viral isolates, we demonstrate that of the five lineages of primate immunodeficiency viruses, only HIV-1 requires cyclophilin A for replication. Cloned viral isolates from clades A, B, and D of HIV-1 group M, as well as a phylogenetically related isolate from chimpanzee, all require cyclophilin A for replication. In contrast, the replication of two outlier (group O) HIV-1 isolates is unaffected by concentrations of cyclosporine A which disrupt cyclophilin A incorporation into virions, indicating that these viruses are capable of replicating independently of cyclophilin A. These studies identify the first phenotypic difference between HIV-1 group M and group O and are consistent with phylogenetic studies suggesting that the two HIV-1 groups were introduced into human populations via separate zoonotic transmission events.