In this investigation we demonstrate that various nucleoside reverse transcriptase inhibitors (NRTIs) and their corresponding nucleotides can cause a direct, DNA polymerase-gamma-independent, inhibition of respiration, membrane potential, and calcium loading capacity in isolated rat heart mitochondria in vitro. Both AZT and d4T also increased total adenine phosphate energy charge in H9c2 rat cardiac myocytes in cell culture. These results demonstrate that the various NRTI nucleosides and nucleotides are capable, at sufficiently high concentrations, of directly affecting mitochondrial bioenergetics in vitro, which may enhance the toxicity observed in vivo previously attributed to inhibition of DNA polymerase-gamma.