Nucleoside analogues can induce mitochondrial dysfunction leading to severe clinical syndromes. Lamivudine, a new nucleoside analogue, is an active inhibitor of hepatitis B viral replication without apparent clinical toxicity. To assess subclinical mitochondrial toxicity, we studied the morphology and function of the mitochondrial system in 15 patients treated with lamivudine. Morphology was investigated by routine histological evaluation and electron-microscopic studies of mitochondria in liver biopsy specimens. Mitochondrial function was assessed by 2-keto[1-14C] isocaproic acid decarboxylation (KICA breath test) and by measuring the activity in liver biopsy specimens of the mitochondrial enzymes encoded by nuclear and mitochondrial DNA (mt-DNA) (complex I and IV) as well as a mitochondrial and a cytosolic enzyme both encoded by nuclear DNA only (complex II and lactic dehydrogenase [LDH]). All 15 patients underwent a liver biopsy before treatment and a KICA breath test before and during treatment; 13 agreed to undergo a repeat liver biopsy during lamivudine treatment. Liver tissue with no or minimal fibrotic changes from 7 patients treated for 6 months with lamivudine was suitable for assessment of the mitochondrial enzyme activity. We observed no signs of toxicity by routine histological or electron-microscopic evaluation. KICA breath tests revealed no differences in either peak exhalation or the area under the curve from 0 to 60 minutes between healthy controls (3.0% and 19.3%), untreated patients with chronic hepatitis B (3.4% and 19.3%), and patients treated with lamivudine (3.1% and 20.6%). The activities of the mt-DNA-encoded enzymes remained normal after lamivudine therapy. Unexpectedly a significant decrease in the activity of nuclear-DNA-encoded enzymes in patients with chronic hepatitis B in comparison with normal controls was found. The mean activity of complex II dropped from 45.3 to 20.0 micromol x min(-1), that of lactic dehydrogenase from 106 to 44 micromol x min(-1) (Wilcoxon rank sum; P < .05). In conclusion, no subclinical signs of mitochondrial toxicity resulting from lamivudine therapy for 6 months were observed.