An improved method for the conjugation of Fab' to B-D-galactosidase fromEscherichia coliwas developed. The enzyme with thiol groups was treated with excess of N, N'-o-phenylene-dimaleimide to introduce maleimide groups, and the maleimide-enzyme was reacted with thiol groups in the hinge of Fab' to form Fab'-B-D-galactosidase conjugate. Gel filtration of Fab' was required only once, and the recovery of Fab' in the conjugate was 71–81%, while in the previous method, gel filtration was required twice and the recovery was 31–38 %. Although the enzyme activity was decreased slightly (8–12%) by the dimaleimide treatment, the conjugate was as useful for sandwich enzyme immunoassay as that obtained by the previous method. 1gG also could be conjugated to B-D-galactosidase more efficiently in the present method than in the previous one.