Little is known about host factors necessary for retroviral virion assembly or uncoating. We have previously shown that the principal structural protein of the human immunodeficiency virus HIV-1, the Gag polyprotein, binds the cyclophilin peptidyl-prolyl isomerases; cyclophilins catalyse a rate-limiting step in protein folding and protect cells from heat shock. Here we demonstrate that cyclophilin A is specifically incorporated into HIV-1 virions but not into virions of other primate immunodeficiency viruses. A proline-rich region conserved in all HIV-1 Gag polyproteins is required for cyclophilin A binding and incorporation. Disruption of a single proline blocks the Gag-cyclophilin interaction in vitro, prevents cyclophilin A incorporation into virions, and inhibits HIV-1 replication. Our results indicate that the interaction of Gag with cyclophilin A is necessary for the formation of infectious HIV-1 virions.