Enzyme immunoassay for cortisol was developed using beta-galactosidase as an enzyme label and m-maleimidobenzoyl derivatives of cortisol, i.e. cortisol-21-m-maleimidobenzoate (CT-MB) and cortisol-21-hemisuccinate conjugated with m-maleimidobenzoic acid through p-phenylenediamine linkage (CHS-MB), as the haptens coupled to sulfhydryl groups of the enzyme. This enzyme-coupling procedure was highly efficient; over 90% of the enzyme was labeled while full enzyme activity was retained. CHS-MB-beta-galactosidase conjugate showed high immunoreactivity to antibody produced against cortisol-21-hemisuccinate-bovine serum albumin but showed poor displacement with the added cortisol. CT-MB-beta-galactosidase conjugate, however, showed not only a high immunoreactivity to the antibody but also displaced well with cortisol, showing maximum sensitivity of 1 microgram/dl with a 20-microliter sample size. Modification around the linkage of cortisol derivative resulted in high sensitivity to cortisol. Cross-reactions to cortisol-21-acetate, cortisone, and corticosterone were 150%, 10%, and 7%, respectively. The accuracy, precision and correlation with RIA of this method were satisfactory for clinical application.