Thermostable enzymes from thermophiles have attracted extensive studies. However, little is known about thermophilic lysin of bacteriophage obtained from deep-sea hydrothermal vent. In this study, a lysin from deep-sea thermophilic bacteriophage Geobacillus virus E2 (GVE2) was characterized for the first time. It was found that the GVE2 lysin was highly homologous with N-acetylmuramoyl-L-alanine amidases. After expression in Escherichia coli, the recombinant GVE2 lysin was purified. The recombinant lysin was active over a range of temperature from 40 degrees C to 80 degrees C, with an optimum at 60 degrees C. Its optimal pH was 6.0, and it was stable over a wide range of pH from 4.0 to 10.0. The lysin was highly active when some enzyme inhibitors or detergents (phenylmethylsulfonyl fluoride, Tween 20, Triton X-100, and chaps) were used. However, it was strongly inhibited by sodium dodecyl sulfate and ethylene diamine tetraacetic acid. Its enzymatic activity could be slightly stimulated in the presence of Na+ and Li+. But the metal ions Mg2+, Ba2+, Zn2+, Fe3+, Ca2+, and Mn2+ at concentrations of 1 or 10 mM showed inhibitions to the lysin activity. Our study demonstrated the first characterization of lysin from deep-sea thermophilic bacteriophage.