Extracts of mitochondria isolated from bovine liver were shown to phosphorylate araG, forming araGMP as the sole product. When other nucleosides were used as competitors with araG as the substrate for phosphorylation, deoxycytidine, deoxythymidine and guanosine were not significantly inhibitory. However, the phosphorylation of araG was blocked by deoxyguanosine, deoxyadenosine and deoxyinosine. Deoxyguanosine was shown to be a competitive inhibitor of araG phosphorylation (apparent Ki for deoxyguanosine = 9 microM; apparent Km for araG = 66 microM). Likewise, araG was determined to be a competitive inhibitor of mitochondrial deoxyguanosine kinase activity (apparent Km for deoxyguanosine = 16 microM; apparent Ki for araG = 55 microM). These data suggest that the two nucleosides were phosphorylated by the same enzyme. Disc gel electrophoresis showed that the phosphorylating activity for araG migrated with deoxyguanosine kinase activity. The pH profiles of the araG and deoxyguanosine kinase activities were dissimilar. The optimum pH for deoxyguanosine kinase was 5.5; for araG kinase, it was 8.0. Collectively, these data suggest that araG is phosphorylated by mitochondrial deoxyguanosine kinase; however, separate forms of the enzyme or different reaction conditions may be involved in the optimal activities of the two catalytic events.