We have discovered that the Mu prophage in Escherichia coli strain DK445, a λcIts857 Sam7 lac5: :Mu cI+ dilysogen (in which Mu is integrated in the lacZ gene of the plac5 prophage in the E. coli chromosome) contains a 2600 base-pair long insertion of unknown nature. The DNAs of seven independent phage Mu isolates recovered from induced DK445 have been studied by electron microscopic heteroduplex methods. All were found to contain either a deletion or a substitution in the G and/or beta regions of the chromosome. All seven mutants have growth rate, burst size and plaque morphology indistinguishable from wild-type Mu. Thus, they are the first non-defective deletion mutants of Mu. The regions deleted in these mutants are non-essential. They span the right-hand 1.3 kilobases of the G segment in the lytic orientation and the entire beta region except the rightmost 0.15 kilobase on the S (right) end. The mechanism which generates these Mu deletion mutants with high efficiency ( 7 7) is not known. The lengths of the bacterial DNA on the S end vary according to the amount of DNA inserted into or deleted from the Mu genome. Therefore, the model of headful encapsidation of Mu DNA from the c (left) end (Bukhari & Taylor, 1975) is substantiated. None of the mutants has lost the right-hand end of beta, as would have been anticipated by straight-forward headful packaging from the c end of prophage with a sizeable insertion. Therefore, the right-hand end of beta, one of its normal attachment sites, is probably essential for Mu viability. The identification of a Mu mutant with a substitution in beta which is incapable of promoting inversion of the G segment suggests that part of the G segment inversion system might be located on the left portion of the beta region. The G segment inverts normally in the Mu prophage in DK445. Yet, all seven deletion mutants have the G segment arranged in the lytic orientation. These observations suggest that the orientation of Mu G DNA may affect the phage viability and that there may be essential functions encoded within the left-hand 1.7 kilobases of the G segment in the lytic orientation. © 1977.