HIV reservoirs are major barriers to the eradication of this infection despite the widespread use of combination antiretroviral therapy. Antiretroviral drugs have restricted entry into the brain due to the inherent complexities of this tissue (e.g. uniquely structured blood-brain barrier, presence of efficient efflux pumps) which makes the brain an ideal potential anatomic reservoir for HIV replication. Cerebrospinal fluid (CSF) and plasma blood concentrations are often used to approximate drug concentrations within brain tissue; however, for reasons previously listed, drug concentrations in these compartments do not necessarily reflect those in the brain. The objective of this study was to evaluate the extent of penetration of select antiretroviral agents into the brain tissue of two preclinical models of HIV infection: rhesus macaques (n=6) and humanized mice (n=12). Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to quantify efavirenz (EFV), emtricitabine (FTC), and tenofovir (TFV) in blood plasma, CSF, and brain tissue. EFV penetrated best into brain tissue in both animal models, and was up to 3.1-fold higher in macaques versus mice (p=0.026). FTC and TFV penetration into brain tissue was 1.3 to 25-fold higher macaques (p=0.004 and p=0.093, respectively). In infected macaques, the CSF-to-plasma ratios (CPRs) for TFV and FTC were 1.6 and 4-fold higher than the brain tissue-to-plasma ratio (TPR). CPRs for the remaining macaque samples were 1.3 to 61-fold lower than the TPRs, suggesting that more drug is getting into the brain tissue compared to the CSF. These results illustrate that CSF drug concentrations may provide underestimations of the penetration of antiretrovirals into brain tissue, and that HIV infection may result in lower brain tissue drug concentrations.