Aminoacylase (EC 3.5.1.14) was immobilized into DEAE-Sephadex A-25 by ion-exchange absorption for optical resolution of N-acyl-DL-alanine. The effects of pH, temperature, and Co2+ concentration on the activity of free and immobilized enzymes were investigated along with the operational and the thermal stability of the immobilized enzyme. The immobilized enzyme retained high catalytic activity. The optimum pH and temperature for the hydrolysis of N-acyl-L-alanine in the DL-isomer mixture were 8.0 and 65 degrees C, respectively. Co2+ was an activator for the immobilized enzyme in a similar role as for the free enzyme. No significant loss of activity was observed for at least 300 h of continuous operation. The yield of L-alanine was about 70% of the theoretical yield. The immobilized aminoacylase column decayed over a very long period of operation, but could be completely reactivated by regeneration.