Nucleoside reverse transcriptase inhibitors (NRTIs) have been used for the last 20 years to fight the progression of the Human Immune Deficiency virus, the virus that causes AIDS. However NRTI antiviral treatments are associated with tissue toxicities that target the mitochondria, decreasing mitochondrial DNA replication. Toxicities associated with NRTIs display significant tissue variability with each NRTI preferentially affecting certain tissues. The mechanisms accounting for this tissue specificity of toxicity is unknown. In previous studies from this laboratory, the effects of NRTIs have been investigated on liver and heart mitochondria, tissues that are associated with known NRTI toxicities. The goal of the project presented here is to expand these studies to brain mitochondria, a tissue relatively resistant to NRTI toxicity. This project has investigated the ability of brain mitochondria to phosphorylate deoxynucleosides and their analogs, and to determine if any of the NRTIs have adverse effects on the phosphorylation of naturally occurring deoxynucleosides. High quality mitochondria were isolated from Harlan Sprague Dawley rat brains and were incubated in media with labeled deoxynucleoside or labeled NRTI to determine the rate and extent of phosphorylation of the deoxynucleoside or analog. In addition, the effects of various NRTIs on rates of phosphorylation of their corresponding deoxynucleosides were investigated. All results were analyzed by HPLC and an in-line scintillation counter. Results demonstrate that the mitochondrial deoxynucleoside salvage pathways are more active in brain mitochondria than in heart or liver mitochondria. Present data show a dramatic increase in the rate of phosphorylation of thymidine (140 pmol/mg) and AZT (140 pmol/mg) individually in brain mitochondria compared to mitochondria from heart (20 pmols/mg and 6 pmol/mg) and liver (40 pmol/mg and 9 pmol/mg) after 90 minute incubations. Phosphorylation of deoxycytidine is also very active with 210 pmol/mg phosphorylated after 180 minutes. Deoxyuridine is phosphorylated to the monophosphate form in brain mitochondria which has also been shown in heart mitochondria, but not in perfused heart. Data demonstrate that AZT is a potent inhibitor of thymidine phosphorylation in brain mitochondria with an IC50 of 5.5