A sensitive and specific enzyme immunoassay for bovine osteocalcin was developed with the use of enzyme-labeled antigen. Bovine osteocalcin was conjugated with beta-D-galactosidase for use in the immunoassay. The minimum amount of osteocalcin detectable by this method was 5-10 pg/assay. The enzyme immunoassay could detect immunoreactive osteocalcin levels in normal human sera, giving a value of 6.58 +/- 0.72 ng/ml (mean +/- SE of 16 human subjects). The correlation coefficient between the levels measured by this method and radioimmunoassay was 0.94.