Methanothermobacter wolfeii (formerly Methanobacterium wolfei), a thermophilic methanoarchaeon whose cultures lyse upon hydrogen starvation, carries a defective prophage called PsiM100 on its chromosome. The nucleotide sequence of PsiM100 and its flanking regions was established and compared to that of the previously sequenced phage PsiM2 of Methanothermobacter marburgensis (formerly Methanobacterium thermoautotrophicum Marburg). The PsiM100 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 PsiM100 but is absent in the genome of PsiM2. The remaining part of the PsiM100 genome showed 70.8% nucleotide sequence identity to the whole genome of PsiM2. Thirty-four open reading frames (ORFs) on the forward strand and one ORF on the reverse strand were identified in the PsiM100 genome. Comparison of PsiM100-encoded ORFs to those encoded by phage PsiM2 and to other known protein sequences permitted the assignment of putative functions to some ORFs. The ORF28 protein of PsiM100 was identified as the previously known autolytic enzyme pseudomurein endoisopeptidase PeiW produced by M. wolfeii.