The role of guanine deaminase in selective cellular resistance to 8-azaguanine was examined, using eight mammalian cell lines and their subclonal derivatives isolated on the basis of increasing resistance to this drug. 8-Azaguanine and 6-thioguanine are synthetic analogs of guanine and are lethal to cells with normal hypoxanthine phosphoribosyltransferase (HPRT) activity. In principle, however, HPRT-positive cells could become selectively resistant to 8-azaguanine if, by any mechanism, the cells expressed higher levels of guanine deaminase. This is because 8-azaguanine, but not 6-thioguanine, is converted by this enzyme to a noncytotoxic metabolite, 8-azaxanthine. Our study shows that HPRT-positive cells inherently resistant to relatively high levels of 8-azaguanine contain high levels of guanine deaminase. In general, guanine deaminase activity was higher in 8-azaguanine-resistant cells, regardless of their HPRT activity. Our results support the view that elevated guanine deaminase activity constitutes a potential mechanism of selective 8-azaguanine resistance in cells with normal HPRT activity. Guanine deaminase levels were significantly elevated in HPRT-positive cells briefly exposed to sublethal concentrations of 8-azaguanine, but this elevation was transient. Long-term exposure of cells to increasingly higher levels of the drug did not lead to high stable levels of guanine deaminase, indicating that 8-azaguanine is not an inducer of guanine deaminase in the cells examined.