The nucleoside analog 2',3'-dideoxycytidine (ddC) has been used for treatment of human immunodeficiency virus (HIV) infections. ddC causes delayed toxicity when cells are exposed to the drug at low concentration for prolonged periods of time. The delayed toxicity is due to inhibition of mitochondrial DNA (mtDNA) replication, which results in mtDNA depletion and mitochondrial dysfunction. In the present study we have cultured CEM T-lymphoblast cells in the presence of low concentrations of ddC to generate two cell lines resistant to the delayed toxicity of the drug. Both cell lines were resistant to mtDNA depletion by ddC. The mechanism of ddC resistance was investigated and we showed that the resistant cells had decreased mRNA expression of the nucleoside kinases deoxycytidine kinase and thymidine kinase 2. We also studied the mitochondrial DNA in the cells and showed that the ddC-resistant cells had structurally intact mtDNA but 1.5-2-fold increased mtDNA copy-number as well as increased levels of the mitochondrial transcription factor A (Tfam). Our study suggests that cells may increase their level of mtDNA to counteract mtDNA depletion induced by ddC, while keeping pronounced antiviral activity of the drug.