To study the activation and cytotoxic mechanism of bredinin (4-carbamoyl-1-β-d-ribofuranosylimidazolium-5-olate), a novel nucleoside antibiotic with potent cytotoxic and immuno-suppressive effects, we isolated in a single-step manner five mutants resistant to 10 μM bredinin from cultured mouse mammary carcinoma FM3A cells mutagenized with N-methyl-N′-nitro-N-nitrosoguanidine (MNNG). Such resistant (Brd^r) mutants were 15- to 19-fold less sensitive to the antibiotic than wild-type cells and maintained stably their resistant phenotypes in the absence of bredinin for more than 3 months. They were cross-resistant to tubercidin, an adenosine analog. Like wild-type cells, Brd^r mutants were capable of incorporating radioactivity from ring-labeled adenosine into the acid-insoluble macromolecular fraction. However, hypoxanthine-guanine phosphoribosyltransferase-deficient (HGPRT^-) mutants derived from the Brd^r cells did not incorporate the radioactivity at all or at a markedly reduced rate, indicating that blockade of the pathway via adenosine deaminase present in the Brd^r cells resulted in loss of their ability to utilize adenosine. Enzyme assays using cell-free extracts revealed that all the Brd^r mutants had less than 3% of the adenosine kinase (AK) activity found in wild-type cells. These results demonstrate that the bredinin resistance is attributed to a defective AK activity and therefore, that bredinin is metabolized by AK, which may phosphorylate it to a toxic nucleotide, bredinin 5'-monophosphate (Brd-MP), in sensitive cells. Among exogenously added purine bases, guanine was able to reverse the cytotoxic effect of bredinin on both wild-type cells and F5 cells carrying the vector pSV2-Escherichia coli xanthine-guanine phosphoribosyltransferase (XGPRT) gene, while xanthine was able to do so only in F5 cells because the base was metabolized to XMP by the cells. These results support the mechanism of bredinin cytotoxicity, that Brd-MP formed in sensitive cells exposed to the antibiotic blocks the conversion of IMP to XMP by inhibiting IMP dehydrogenase.