A sensitive enzyme immunoassay for plasma betamethasone was developed using betamethasone-3-(O-carboxymethyl)oxime-beta-D-galactosidase conjugate as a labelled antigen and 4-methylumbelliferyl-beta-D-galactoside as a fluorescence substrate. The performances of the enzyme immunoassay were compared with that of a radioimmunoassay using 3H-betamethasone and the same antiserum. The minimal detectable level for the enzyme immunoassay was 0.15 pg/tube or 0.15 ng/ml of plasma, which was remarkably more sensitive than the radioimmunoassay level of 10 pg/tube or 2 ng/ml of plasma. The specificity was sufficient, in particular, the cross reactivity of cortisol as 0.008%. However, the precision of the enzyme immunoassay was inferior to that of the radioimmunoassay.