While the human antiretroviral defense factors APOBEC3F and APOBEC3G are potent inhibitors of the replication of HIV-1 mutants lacking a functional vif gene, the Vif protein expressed by wild-type HIV-1 blocks the function of both host cell proteins. Here, we report that a third human protein, APOBEC3B, is able to suppress the infectivity of both Vif-deficient and wild-type HIV-1 with equal efficiency. APOBEC3B, which shows approximately 58% sequence identity to both APOBEC3F and APOBEC3G, shares the ability of these other human proteins to bind the nucleocapsid domain of HIV-1 Gag specifically and to thereby package into progeny virion particles. However, APOBEC3B differs from APOBEC3F and APOBEC3G in that it is unable to bind to HIV-1 Vif in co-expressing cells and is therefore efficiently packaged into HIV-1 virions regardless of Vif expression. Unfortunately, APOBEC3B also differs from APOBEC3F and APOBEC3G in that it is not normally expressed in the lymphoid cells that serve as targets for HIV-1 infection. These studies therefore raise the possibility that activation of the endogenous APOBEC3B gene in primary human lymphoid cells could form a novel and effective strategy for inhibition of HIV-1 replication in vivo.