This project defines in vivo mechanisms of acquired mitochondria! (mt-) DNA depletion caused by nucleoside reverse transcriptase inhibitors (NRTI) for HIV/AIDS. Survival with HIV/AIDS improved because of pyrimidine NRTIs like zidovudine (AZT) and stavudine (d4T), but NRTI mitochondrial toxicity limits therapy and mani- fests as mtDNA depletion and organ dysfunction. The "DNA pol y hypothesis" underscores NRTI phosphor- ylation (to active moieties) and inhibition of DNA pol y (the mtDNA replicase) by NRTI triphosphates (-TP) in the mechanism of acquired mtDNA depletion. Cellular kinases control mitochondrial nucleotide and NRTI phosphorylations. Pyrimidine NRTIs compete with dThd for phosphorylation to monophosphates (-MP) by thymidine kinase (cytoplasmic TK1 and mitochondrial TK2 isoforms) and to diphosphates (-DP) by thymidylate kinase (TMPK). The working hypothesis states: Transgenic (TG) over-expression of wild type (WT) TK2 or WT TMPK promotes mtDNA replication by increasing the mitochondrial dTTP mass in parallel with increased enzyme abundance. TGs with TMPK mutants ("gain of function") are active with dTMP phosphorylation, but exhibit enhanced activity with AZTMP. In absence of AZT, these TGs each increases dTTP abundance. With AZT therapy, AZT-DP abundance increases dramatically and is imported into mito- chondria for phosphorylation to AZT-TP. Conversely, TGs harboring specific TK2 point mutations decrease TK2 activity ("loss of function"). These latter TGs deplete mtDNA by limiting mitochondrial dTMP for down- stream intra-mitochondrial processing. AZT treatment here enhances mtDNA depletion by AZT's competition with dThd for phosphorylation by TK2. Mitochondrial AZT-TP competes with dTTP for incorporation into mtDNA by DNA pol y. This inhibits mtDNA replication, causes mtDNA depletion and mutation, mitochondrial dysfunction, and organ dysfunction. AIM 1 defines molecular, pathological and physiological events in mtDNA depletion from NRTIs (like AZT) using TGs with cardiac targeted, conditional, overexpression of WT TMPK, chimeric TMPK, and mutant TMPK. AIM 2 defines molecular, pathological, and physiological events in mtDNA depletion from NRTIs (like AZT) using TGs with cardiac targeted, conditional, overexpressionof WT TK2 and mutant TK2. Experiments offer insights into improving therapy with antiretrovirals used in AIDS.