Abstract Enzyme immunoassay of corticosterone was developed using alkaline phosphatase as a labelling enzyme. Serum corticosterone was measured by this system after the extraction and purification of corticosterone by Sephadex LH-20 column chromatography. The sensitivity of this assay was 50 pg and the measurable range was from 0.02 μg/dl to 5.0 μg/dl. Intra- and inter-assay coefficient of variation were 12% and 20%, respectively. Corticosterone values determined by the present method correlated well with those determined by radioimmunoassay. Normal corticosterone levels in 20 healthy subjects at 9.30 a.m. were 0.24 ± 0.09 μg/dl. This enzyme immunoassay satisfied the standard criteria of dilution, accuracy and precision, and is applicable to routine determination of serum corticosterone in any clinical laboratory.