A lytic phage, designated as ϕTMA, was isolated from a Japanese hot spring using Thermus thermophilus HB27 as an indicator strain. Electron microscopic examination showed that ϕTMA had an icosahedral head and a contractile tail. The circular double-stranded DNA sequence of ϕTMA was 151,483 bp in length, and its organization was essentially same as that of ϕYS40 except that the ϕTMA genome contained genes for a pair of transposase and resolvase, and a gene for a serine to asparagine substituted ortholog of the protein involved in the initiation of the ϕYS40 genomic DNA synthesis. The different host specificities of ϕTMA and ϕYS40 could be explained by the sequence differences in the C-terminal regions of their distal tail fiber proteins. The ΔpilA knockout strains of T. thermophilus showed simultaneous loss of sensitivity to their cognate phages, pilus structure, twitching motility and competence for natural transformation, thus suggesting that the phage infection required the intact host pili. Pulsed-field gel electrophoresis analysis of the ϕTMA and ϕYS40 genomes revealed that the length of their DNA exceeded 200 kb, indicating that the terminal redundancy is more than 30% of the closed circular form. Proteomic analysis of the ϕTMA virion using a combination of N-terminal sequencing and mass spectrometric analysis of peptide fragments suggested that the maturation of several proteins involved in the phage assembly process was mediated by a trypsin-like protease. The gene order of the phage structural proteins was also discussed.