Although treatments with nucleoside reverse transcriptase inhibitors (NRTIs) can modify fat metabolism and fat distribution in humans, the mechanisms of these modifications and the roles of diverse NRTIs are unknown. We studied the mitochondrial and metabolic effects of stavudine (d4T), zidovudine (AZT), didanosine (ddI), lamivudine (3TC), zalcitabine (ddC), and three combinations (AZT-3TC, d4T-3TC, and d4T-ddI) in mice treated for 2 weeks with daily doses equivalent to the human dose per body area. Concentrations of AZT and d4T in plasma were lower when these drugs were administered with 3TC or ddI. Whatever the treatment, mitochondrial DNA was not significantly decreased in muscle, heart, brain, or white adipose tissue but was moderately decreased in liver tissue after the administration of AZT, 3TC, or d4T alone. Blood lactate was unchanged, even when NRTIs were administered at supratherapeutic doses. In contrast, the level of plasma ketone bodies increased with the administration of AZT or high doses of d4T but not with ddC, 3TC, or ddI, suggesting that the thymine moiety could be involved. Indeed, the levels of plasma ketone bodies increased in mice treated with beta-aminoisobutyric acid, a thymine catabolite. Treatment with AZT, d4T, or beta-aminoisobutyric acid increased hepatic carnitine palmitoyltransferase I (CPT-I) mRNA expression and the mitochondrial generation of ketone bodies from palmitate. In conclusion, therapeutic doses of NRTIs have no or moderate effects on mitochondrial DNA and no effects on plasma lactate in mice. However, AZT and high doses of d4T increase the levels of hepatic CPT-I, mitochondrial fatty acid beta-oxidation, and ketone bodies, and these catabolic effects are reproduced by beta-aminoisobutyric acid, a thymine metabolite.