Using nondegradative isolation procedures, we purified and characterized a glycoprotein from fetal calf bone that is rich in sialic acid. This bone sialoprotein (BSP) has an apparent Mr = 70,000-80,000 and stains with Alcian blue and Stains All on sodium dodecyl sulfate gels but does not stain with Coomassie blue without prior treatment with neuraminidase. This glycoprotein contains 50% protein, 12% sialic acid, 7% glucosamine, and 6% galactosamine. Fetal calf BSP is rich in glutamate (19%), aspartate (15.4%), and glycine (11.8%) but, in contrast to osteonectin and the bone proteoglycan, has relatively low amounts of leucine (4.3%). Antisera raised against fetal calf BSP localized the glycoprotein by indirect immunofluorescence to developing bone trabeculae with an overall tissue distribution identical with that of osteonectin. On competition enzyme-linked immunosorbent assay analysis, BSP was 11.5% (+/-2.4%, S.E.) of mineral-bound (guanidine-EDTA-soluble) calf bone protein. Immunoreplicas (Western blots) of calf bone extracts suggest that more than 95% of the antigenicity resided in the Mr = 70,000-80,000 region with the remaining cross-reactivity in Alcian blue positive, Mr = approximately 20,000 and approximately 30,000 bands. Brief treatment of the Mr = 70,000-80,000 species with trypsin produced lower molecular weight, Alcian blue-staining products of similar size. No BSP was detected in guanidine extracts of various soft or unmineralized connective tissues, but dentin contained small amounts (0.4%) of the protein. Rat and fetal human bone were also observed to contain a sialoprotein with similar properties and a certain degree of cross-reactivity with the bovine BSP.