Mutants of Escherichia coli which are resistant to 5-fluorodeoxyuridine all have mutations which map at a single locus at 27.5 min on the genetic map of E. coli. Extracts prepared from each mutant were deficient in thymidine kinase activity measured in vitro. Simple selective conditions which allowed detection of one mutant in the presence of 10(7) wild-type bacteria were found. These results show that loss of thymidine kinase activity is the usual mechanism for 5-fluorodeoxyuridine resistance and that all such mutations occur at the locus previously designated tdk.