SUMMARY: Two virulent, archaeal phages, øF1 and øF3, were isolated that were capable of infecting different thermophilic members of the genus Methanobacterium. Both phages exhibited a similar morphology consisting of a polyhedral head and a tail but differed considerably in their host specificities and the size and topology of their genomes. Phage øF1 contained a linear, double-stranded DNA genome of 85 + 5 kb in size and showed a broad host range including M. thermoformicicum strains Z-245, FTF, FF1, FF3 and CSM3, and M. thermoautotrophicum strain ΔH. In contrast, øF3 phage particles contained a circular or terminally redundant linear genome, comprising approximately 36 + 2 kb double-stranded DNA, and could only be propagated on M. thermoformicicum strain FF3. Hybridization experiments did not reveal similarity between the genomes of øF1 and øF3 nor between both phages and genomic DNA from different thermophilic members of the genus Methanobacterium or DNA from phage øM1 of M. thermoautotrophicum Marburg. A physical map of both phage genomes was constructed. The DNA of phage øF1 was found to contain multiple GGCC sites which form the target for the restriction-modification (R/M) system MthTI of M. thermoformicicum THF. In contrast, the DNA of øF1 contained only a single CTAG site recognized by the R/M systems MthZI and MthFI of M. thermoformicicum Z-245 and FTF, respectively. The distribution of these sites correlates well with the capacity of øF1 to infect M. thermoformicicum strains Z-245 and FTF but not strain THF.