The main carbohydrate of red macroalgae is agarose, a heterogeneous polysaccharide composed of d-galactose and 3,6-anhydro-l-galactose. When saccharifying agarose by enzymes, the unique physical properties of agarose, namely the sol-gel transition and the near-insolubility of agarose in water, limit the accessibility of agarose to the enzymes. Due to the lower accessibility of agarose to enzymes in the gel state than to the sol state, it is important to prevent the sol-gel transition by performing the enzymatic liquefaction of agarose at a temperature higher than the sol-gel transition temperature of agarose. In this study, a thermostable endo-type beta-agarase, Aga16B, originating from Saccharophagus degradans 2-40(T), was characterized and introduced in the liquefaction process. Aga16B was thermostable up to 50 A degrees C and depolymerized agarose mainly into neoagarooligosaccharides with degrees of polymerization 4 and 6. Aga16B was applied to enzymatic liquefaction of agarose at 45 A degrees C, which was above the sol-gel transition temperature of 1 % (w/v) agarose (similar to 35 A degrees C) when cooling agarose. This is the first systematic demonstration of enzymatic liquefaction of agarose, enabled by determining the sol-gel temperature of agarose under specific conditions and by characterizing the thermostability of an endo-type beta-agarase.