Maltose phosphorylase (MP) and trehalose phosphorylase (TP) in the crude extract from a strain of Plesiomonas were adsorbed simultaneously on an anion-exchange resin to examine the continuous production of trehalose from maltose. The activity recoveries of both enzymes after immobilization were about 97% for MP, 33% for TP, and 39% for the trehalose-forming activity (TFA). The immobilized enzyme(s) expressed by TFA was stable at nearly neutral pH and showed about 96% of the original activity after a 1-h incubation at 55°C and pH 7.0 after which the nonimmobilized enzymes completely lost their activities. About 10 m or more of inorganic phosphate (Pi) was required for TFA of the immobilized enzyme to achieve its full activity, and apparent values of the Michaelis constant (Km) and maximum velocity (Vmax) for Pi were about 0.2 m and 94.2 μmol trehalose min(-1) mg(-1) protein at pH 6.2 and 50°C, respectively. The maximum yield of trehalose from maltose after removing small amounts of glucose-1-phosphate (G-1-P) which was formed concomitantly during the reaction was about 60%. The optimum flow rate achieving the maximum productivity of trehalose was about 1.0 h(-1) of the space velocity (SV) at 20% (w/v) maltose concentration at which the productivity was 252 mg trehalose h(-1) g(-1). Half-lives of the immobilized enzyme in continuous operation at 0.2 h(-1) of SV and pH 6.2 in the presence of 10 m Pi with 30% (w/v) corn syrup containing about 84% as dry basis of maltose as a substrate were calculated at about 164 days at 55°C, 28 days at 60°C, and 8 days at 65°C, respectively. Using the method reported here, the mother liquor for preparing crystalline trehalose from maltose can be produced conveniently on an industrial scale.