Nucleoside reverse transcriptase inhibitors (NRTIs) have been hypothesized to inhibit mitochondrial DNA polymerase gamma, resulting in decreased mtDNA synthesis and mitochondrial insufficiency in HIV-1-infected patients. mtDNA synthesis was measured directly using a stable isotope mass spectrometric method following NRTI treatment in rodents. 3'-Azido-3'-deoxythymidine (AZT) was added to water (1 mg/mL) and administered ad libitum to female Sprague-Dawley rats for 1-8 weeks (n = 4 or 5 animals/timepoint). Neither body weight nor food intake was affected by AZT intake. Untreated controls and AZT-treated rats were given 4% H2O as drinking water for 2 weeks. AZT (approximately 100 mg/kg/d) produced a significant (P < 0.05) decrease in cardiac and hindlimb muscle mtDNA fractional synthesis compared with control groups (from 13.8 +/- 4.2% to 7.0 +/- 4.8% and from 7.6 +/- 1.8% to 4.5 +/- 0.4%, respectively) after 4 weeks. Cytochrome c oxidase content in hindlimb muscle was also decreased by 50% compared with controls after 4 weeks of AZT treatment (P < 0.07) and a calculated index of absolute mitochondrial biogenesis rate was significantly reduced by week 2 of AZT (P < 0.05) in hindlimb muscle. In preliminary studies, platelet mtDNA enrichments were compared to monocyte nDNA enrichments (used as a marker of a fully turned over tissue) in healthy human subjects. Fractional synthesis of mtDNA in platelets reached 98 +/- 3% after 5 weeks of H2O labeling. It is concluded that NRTIs decrease mtDNA synthesis and oxidative enzyme content and thus mitochondrial biogenesis in rodents and that the effects of NRTIs on mitochondrial biogenesis in tissues of HIV-1- infected humans can in principle be measured using this approach.