Several studies have shown that deletion of the nef gene of simian immunodeficiency virus (SIV) and simian-human immunodeficiency virus (SHIV) results in attenuated viruses. However, studies have not critically examined trafficking of attenuated viruses to the central nervous system (CNS) at early stages after inoculation. In this study, we investigated the colocalization of pathogenic and vpu-negative, nef-interrupted SHIVs at early stages following inoculation. The first virus, designated SHIV(50OLNV), was isolated from the lymph node of a pig-tailed macaque which developed severe CD4+ T cell loss and neurological disease. The second virus was a molecularly cloned virus in which the vpu gene was deleted and the gene for the enhanced green fluorescent protein from the jellyfish Aequoria victora had been inserted in-frame within the nef gene of the pathogenic SHIV(KU-1bMC33) (designated SHIV(KU-1bEGFP)). Three pig-tailed macaques were inoculated intravenously with equivalent amounts of two viruses, two macaques were inoculated with SHIV(KU-1bEGFP), and two macaques were inoculated with SHIV(50OLNV). The peripheral blood mononuclear cells (PBMCs) were isolated from bleeds obtained 3, 7, 10, and 14 days postinoculation and monitored for syncytia-inducing virus and for fluorescent cells. Virus was detected in the PBMCs as early as 3 days postinoculation and was present throughout the course of this short-term study. At 14 days postinoculation, the macaques were sacrificed and examined for virus in lymphoid tissues and different regions of the CNS following necropsy. Our results revealed the presence of both viruses in lymphoid and CNS tissues, although SHIV(50OLNV) was present to a much greater extent. Histological examination revealed that one macaque displayed signs of meningitis and all three macaques developed massive cortical astrocyte activation as demonstrated by immunostaining for glial fibrillary acidic protein, but only limited microglial activation. In the two macaques inoculated with SHIV(50OLNV), astrocyte activation similar to that in the macaques inoculated with both viruses was observed while no astrocyte activation was observed in macaques inoculated with SHIV(KU-1bEGFP). Thus, this study demonstrates that SHIVs with an intact nef(SHIV(50OLNV)) as well as those lacking a vpu gene and with a nonfunctional nef gene (SHIV(KU-1bEGFP)) are capable of invading the CNS and that pathogenic SHIVs are capable of causing reactive astrocytosis early after inoculation.