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This study describes for the first time the development and validation of a sensitive and specific radioimmunoassay (RIA) for equine osteocalcin (OC) quantification using purified equine OC as standard, tracer, and immunogen for antibody formation in rabbits. The assay allowed to measure equine serum OC levels with a sensitivity of 0.2 ng/mL. Immunoreactive serum OC values of clinically normal, different-aged horses ranged from 3.68 to 127.31 ng/mL. Intra- and inter-assay coefficients of variation (CV) were 6.2 and 8.2%, respectively. Serial equine serum sample dilutions were linear. The recovery of equine OC from equine serum samples ranged from 93.88 to 107.9%. There was a tight correlation between OC values measured with the equine-specific OC RIA and two commercially available bovine-specific OC RIA kits. However, highest serum OC values were obtained with the equine-specific OC RIA. In conclusion, our equine-specific OC RIA is sensitive, linear, accurate, precise, and reproducible. The assay allowed to quantify OC in equine serum samples and might, therefore, be used to monitor equine osteoblast activity associated with bone diseases, exercise, therapy forms or diet.