By selection for resistance to fluorodeoxyuridine in a fluorouracil/fluorouridine resistant background, we have isolated a thymidine kinase negative mutant of Nocardia lactamdurans. This strain is characterized by the inability to incorporate exogenous [2-14C]-thymidine into DNA. The incorporation of radioactive thymine is similarly reduced even in the presence of deoxyadenosine. This phenotype is readily explained by the inability to detect the enzyme thymidine kinase in crude extracts.