Nucleoside analog reverse transcriptase inhibitors (NRTIs) constitute the most commonly used drugs in antiretroviral therapy. NRTIs differ with respect to their host cell toxicity. We compared the in vitro effect of zidovudine (AZT; 2 μg/ml), lamivudine (3TC; 5 μg/ml), stavudine (d4T; 1 μg/ml), and tenofovir (TFV; 1 μg/ml) on Candida cell-mediated immunity (CMI) of peripheral blood mononuclear cells (PBMCs). The concentrations of the active derivative AZT-triphosphate were 4-fold higher in Candida-stimulated compared with unstimulated PBMCs (p = 0.01), but those of 3TC-triphosphate and TFV-diphosphate did not differ significantly. AZT treatment decreased proliferation of unstimulated and Candida-stimulated PBMCs and IFN-γ ELISPOT responses; 3TC decreased proliferation of unstimulated PBMCs only; d4T and TFV decreased proliferation of Candida-stimulated PBMCs only. AZT, but not the other NRTIs, increased unstimulated PBMC apoptosis measured by caspase 3 activity. All NRTIs increased annexin-V-measured apoptosis of Candida-stimulated PBMCs. The effect of d4T on apoptosis of Candida-stimulated PBMCs strongly correlated with its inhibitory effect on mitochondrial DNA synthesis (r² = 0.94; p = 0.007). The other NRTIs did not significantly decrease the mitochondrial:nuclear DNA ratios in Candida-stimulated or unstimulated cultures, suggesting that other mechanisms mediated their effect on apoptosis and CMI. In conclusion, AZT had the most pronounced inhibitory effect on CMI. Further studies are warranted to determine the clinical significance of this observation.