Enzyme labeling of steroids by the p-nitrophenyl ester method was investigated in comparison with the N-succinimidyl ester method. The active ester of a testosterone or 11-deoxycortisol derivative was treated with beta-galactosidase and horseradish peroxidase to give labeled antigens. Various molar ratios of steroid to enzyme and pH conditions were tested. Satisfactory immunoreactivities with an anti-steroid antibody in each enzyme immunoassay system were obtained with the labeled antigens prepared at pH 8.5 by the use of molar ratios higher than 30. The enzyme labeling method should be useful in the case of polar steroids or drugs, since the p-nitrophenyl ester is relatively stable when compared with the N-succinimidyl ester.