Simian immunodeficiency virus (SIV) infection of macaques results in neurological abnormalities similar to those of human immunodeficiency virus (HIV)-associated dementia in humans and is a valuable system for the identification of viral neurotropic and neurovirulence factors. The authors recently established an SIV-macaque model where macaques can be infected with wild-type or nef/LTR-deleted SIVmac239 via administration of purified proviral DNA. In this study, the ability of wild-type and nef/LTR-deleted SIV infections to enter the cerebral spinal fluid (CSF) and brain was analyzed. In situ polymerase chain reaction (PCR) readily detected SIV gag DNA-positive cells in the mid-frontal gyrus and basal ganglia of the wild-type SIV-infected macaques, but not in nef/LTR-deleted SIV-infected or SIV-uninfected macaques. PCR on extracted DNA confirmed the in situ results, with multiple brain regions of the wild-type SIV-infected macaques positive for both gag and wild-type nef, whereas in the nef/LTR-deleted SIV-infected macaques, nef/LTR and gag DNA were undetectable. Further, macaques infected with nef/LTR-deleted SIV, which later became superinfected with wild-type SIV, also remained negative for SIV DNA in the brain by both in situ and extracted DNA techniques, despite having high levels of SIV RNA both in the CSF and plasma. This study provides evidence of the inability of nef/LTR-deleted SIV to initiate central nervous system (CNS) infection and suggests that, in the brain regions examined, nef/LTR-deleted viruses have either diminished neurotropism or insufficient systemic viral replication for entry into the CNS.