Natural hosts of SIV, such as sooty mangabeys (SM), maintain high levels of virus replication, but do not typically develop CD4+ T cell loss and immunodeficiency. Understanding the virus/host relationship in natural hosts will enable better understanding of pathogenic HIV infection of humans. Host cell targeting in vivo is an important determinant of pathogenesis, and is defined mainly by expression of coreceptors used by the virus for entry, in conjunction with CD4. Established dogma holds that, with rare exceptions, SIV uses CCR5 for entry. However, SM and other natural hosts express extremely low CCR5 levels on CD4+ T cells. I identified a novel SM-CCR5 mutant allele containing a two base-pair deletion (CCR5-d2) that results in a non-functional protein. Using PCR screening and direct sequence confirmation in a large captive SM colony, I found an allelic frequency of 26% for CCR5-d2, along with 3% for a previously-described CCR5-d24 null allele. Notably, SM-CCR5-d2 was also present in West African wild-living SM. Approximately 8% of captive SM were homozygous for CCR5-null alleles. Surprisingly, SIVsmm infection was not significantly less prevalent in SM lacking functional CCR5 compared to CCR5-expressing animals, and CCR5-null animals displayed high-level viremia. Primary PBMC from SM were permissive for SIVsmm replication in both the absence and presence of CCR5 in vitro. Pseudotype virions carrying Envs from both CCR5-null and wild-type infected SM used CXCR6 and GPR15 efficiently for entry in transfected cells, suggesting likely alternative entry molecules. In preliminary studies using human cells, I found that CD4+ T cell expression of CXCR6 and GPR15 was predominantly on central and effector memory subsets, although expression patterns on SM cells will require antibodies that can detect SM proteins. This finding challenges the long-standing notion that CCR5 is the exclusive coreceptor used for SIV entry in vivo, and reveals that SIVsmm can infect target cells in vivo using non-CCR5 entry pathways. Since CD4/coreceptor interactions determine viral tropism and cell targeting, these results suggest that infection in natural host SM may involve target cells defined by the distribution and use of alternative coreceptors CXCR6 and GPR15. Combined with restricted expression of CCR5 on critical target cells previously described, alternative coreceptor-dependent targeting may allow virus replication in more expendable cells without loss of immune cell homeostasis. We hypothesize that SM natural hosts may have evolved to restrict expression of CXCR6 and GPR15, in addition to CCR5, to dispensable cell subsets, thus allowing for high viral replication without causing disease. Future studies comparing CXCR6 and GPR15 expression profiles in natural and non-natural hosts will be of importance in determining the role of alternative coreceptors in natural hosts in vivo.