Mutants of bacteriophage T5 were isolated which lack one or more of the natural single-chain interruptions that occur in the mature DNA of this virus. Interruption-deficient mutants were detected by screening survivors of hydroxylamine mutagenesis for altered DNA structure by electrophoresis in agarose slab gels. Over 60 independent mutants were isolated from a survey of approximately 800 phage particles. All of the mutants were viable and could be grouped into two classes. Mutants in one class lacked one of the localized sites where interruptions occur in T5 DNA. To date, mutants that affect five different sites have been obtained. Mutants in the other class were essentially free from interruptions or had a reduced frequency of interruptions throughout the genome. The members of this class included several amber mutants. Complementation tests indicated that at least two genes are required for the presence of interruptions in mature T5 DNA.