Fosalvudine tidoxil is a prodrug derived from the nucleoside reverse transcriptase inhibitor 3-deoxy-3-fluorothymidine (FLT; alovudine). FLT effectively inhibits resistant human immunodeficiency virus type 1, but its clinical development was stopped due to bone marrow and liver toxicity. In this study, we examined the long-term in vivo effects of fosalvudine tidoxil on the mitochondrial DNA (mtDNA) contents in rats. Sprague-Dawley rats received fosalvudine tidoxil (15, 40, or 100 mg/kg of body weight/day) by oral gavage during a period of 8 weeks. Didanosine (100 mg/kg/day) was used as a positive control for mitochondrial toxicity. mtDNA levels in liver, gastrocnemius muscle, sciatic nerve, and inguinal fat pad tissues were quantified by real-time PCR. In hepatic mitochondria, fosalvudine tidoxil induced significant mtDNA depletion. At doses of 15, 40, and 100 mg/kg, the mean hepatic mtDNA values were 62, 64, and 47% of control values, respectively. Rats exposed to 100 mg/kg of fosalvudine tidoxil, unlike all other groups, had slightly elevated levels of glutamate pyruvate transaminase in sera. Didanosine induced a loss of mtDNA (to 48% of the control level) similar to that induced by fosalvudine tidoxil. mtDNA levels in skeletal, neural, and adipose tissues in the negative control and treatment groups were similar. Our results suggest that fosalvudine tidoxil induces mitochondrial hepatotoxicity and that this effect warrants scrutiny in clinical trials.