Insulin-like growth factor binding protein-3 (IGFBP-3), the most prominent IGF-binding protein in serum, has been demonstrated to modulate the effects of the IGFs but also to exert IGF-independent actions. Quantification of IGFBP-3 in livestock species, in particular ruminants, is commonly limited to blotting methods in spite of the importance of these species. Here we describe the development of a specific homologous enzyme-linked immunosorbent assay (ELISA) to measure bovine IGFBP-3 in bovine plasma, serum and milk. IGFBP-3 purified from bovine serum was used both as standard and also for tracer synthesis. A specific antiserum was raised in rabbits using a synthetic peptide based on the sequence of bovine IGFBP-3. The measuring range of the assay was between 50 and 1000 ng IGFBP-3 per milliliter of plasma or milk. Mean recovery was 97.3% for plasma and 100.1% for milk. Intra- and interassay coefficients of variation were 6.2% and 9.3%, respectively. For the biological verification of the assay, IGFBP-3 was determined in plasma obtained from 12 dairy cows before and after being injected with a depot-formulated growth hormone (GH) preparation. GH, a well-characterized stimulator of IGFBP-3, led to a 1.3-fold increase of basal IGFBP-3 concentrations during days 3 to 19 after the injection. The availability of an ELISA procedure which permits precise and sufficiently sensitive quantification of bovine IGFBP-3 and which can be used on large sample numbers thereby avoiding the need for radioactive labels, should facilitate further research studies.