An efficient procedure for the conjugation of rabbit Fab' with beta-D-galactosidase from Escherichia coli using N,N-o-phenylenedimaleimide is described. Thiol groups of Fab' were stabilized by the presence of ethylenediaminetetraacetate, and malemide groups were shown to be stable at pH 5 at 4 degrees C. The stability of thiol and maleimide groups enabled an efficient introduction of maleimide groups into Fab' and the average number of maleimide groups introduced into Fab' was 0.76 (range 0.73-0.79; n = 10) per molecule. As a result, 43.4% (range 41.3-46.9%; n = 6) of Fab' used could be conjugated with most of beta-D-galactosidase used. The average number of Fab' molecules conjugated per enzyme molecule was calculated to be 4.2 (range 4.0-4.6; n = 6). Both the enzyme and antibody activities were well preserved in the conjugate. There was no self-coupling of Fab', although the enzyme was polymerized to some extent during the conjugation reaction. The enzyme activity and cross-link in the conjugate was stable at pH 6.0-7.0 at 4 degrees C for at least 3 months.