Primary isolates of human and simian immunodeficiency viruses (HIV and SIV) use the chemokine receptor CCR5, in association with CD4, as coreceptor. During AIDS progression, HIV-1 and HIV-2 often adapt to use additional cofactors, particularly CXCR4. In contrast, SIV isolates do not use CXCR4, but other coreceptors such as BOB/GPR15 and Bonzo/STRL33. Only limited information is currently available on usage of BOB/GPR15 and Bonzo/STRL33 by HIV-1. Therefore, we investigated a panel of gp160 clones from 15 primary isolates, representing 5 different subtypes, for utilization of these cofactors. The majority of HIV-1 envelopes mediated entry into BOB/GPR15-expressing cells, albeit often with low efficiency. Usage of Bonzo/STRL33 was less common and usually inefficient. To investigate if HIV-1 entry via these orphan receptors is sufficient to allow virus replication, 15 uncloned primary HIV-1 isolates and 7 molecular clones were used to infect target cells expressing CD4 and Bonzo/STRL33 or BOB/GPR15. Three primary isolates and two molecular clones replicated efficiently in cells expressing BOB/GPR15. Two of these isolates were X4-tropic, two were R5X4-tropic and one was R5-tropic. In contrast, none of the HIV-1 variants showed significant levels of replication in Bonzo/STRL33-expressing cells. Our data show that some HIV-1 isolates of different genetic subtype and of different biological phenotype use BOB/GPR15 for productive infection and suggest that this cofactor may play a role in HIV-1 pathogenesis and transmission.