Methanothermobacter wolfeii (formerly Methanobacterium wolfei), a thermophilic methanoarchaeon whose cultures lyse upon hydrogen starvation, carries a defective prophage called M100 on its chromosome. The nucleotide sequence of M100 and its flanking regions was established and compared to that of the previously sequenced phage M2 of Methanothermobacter marburgensis (formerly Methanobacterium thermoautotrophicum Marburg). The M100 genome extends over 28,798 bp, and its borders are defined by flanking 21-bp direct repeats of a pure-AT sequence, which very likely forms the core of the putative attachment site where the crossing over occurred during integration. A large fragment of 2,793 bp, IFa, apparently inserted into M100 but is absent in the genome of M2. The remaining part of the M100 genome showed 70.8% nucleotide sequence identity to the whole genome of M2. Thirty-four open reading frames (ORFs) on the forward strand and one ORF on the reverse strand were identified in the M100 genome. Comparison of M100-encoded ORFs to those encoded by phage M2 and to other known protein sequences permitted the assignment of putative functions to some ORFs. The ORF28 protein of M100 was identified as the previously known autolytic enzyme pseudomurein endoisopeptidase PeiW produced by M. wolfeii.