A highly specific procedure for the isolation of deletion mutants is described. The size and location of the deletions can be predetermined. By this method a series of deletion mutants mapping within and near the untranslated 5' leader sequence of the late 16S mRNA of simian virus 40 have been isolated. The boundaries of the deletions have been accurately determined by DNA sequence analysis. The deletions range from 20 to 223 nucleotides. All these deletions mutants are viable and grow without helper virus. The largest of these deletions removes the entire leader sequence except for six nucleotides at the 3' end that are probably involved in covalent linkage with the 5' end of the body of the mRNA located 937 nucleotides away on the genome. Three of the deletion mutants remove the 5' end of the leader that normally bears the cap structure of the mRNA. A large segment immediately preceding the leader sequence is also removed in one of these mutants, ruling out the generation of the 5' end of the mRNA via initiation of transcription at this point. The circularization of linear infecting DNA producing the DNA of the deletion mutants proceeds mainly by way of blunt end ligation in vivo.