Background: The metabolism of pyrimidine deoxynucleosides and nucleoside reverse transcriptase inhibitors has been studied in growing cells. However, many of these drugs are associated with mitochondrial toxicities observed in nonreplicating tissues, such as in the heart, where their metabolism has not been investigated. Methods: The aims of this study were twofold. The first was to investigate the metabolism of the thymidine analogues 3′-azido-3′ deoxythymidine (AZT) and 2′,3′-didehydrodideoxy-thymidine (d4T), and the deoxycytidine (dCyd) analogues 2′-deoxy-3′ thiacytidine (3TC) and 2′,3′-dideoxycytidine (ddC) with regard to phosphorylation and breakdown. The second was to investigate their potential effects, singly or in combination with AZT, on metabolism of the naturally occurring deoxynucleosides in the perfused rat heart and in isolated heart mitochondria. Results: The analogue d4T was not metabolized in perfused heart or in isolated mitochondria, and had no effect on either thymidine or dCyd metabolism. The dCyd analogues were both phosphorylated in perfused heart to the triphosphate, but only at the limit of detection and they were not phosphorylated in isolated mitochondria. Neither ddC nor 3TC had any effect on thymidine or dCyd metabolism in either perfused heart or in isolated mitochondria. AZT has been previously shown to inhibit thymidine phosphorylation. When d4T, 3TC or ddC were given with AZT, only ddC caused a significant further decrease in thymidine phosphorylation. Conclusions: These results indicate that with the exception of the competition between AZT and thymidine, there was little competition for phosphorylation among and between these other nucleoside reverse transcriptase inhibitors and the naturally occurring deoxynucleosides in cardiac tissue and isolated heart mitochondria.