Bovine serum is a constituent of most media used for the culture of animal cells. The adhesion-promoting properties of serum are generally attributed to fibronectin, yet there have been frequent reports of other adhesion-promoting molecules in bovine serum. Using a technique in which adhesive proteins are visualized after separation by SDS-PAGE, we graphically confirm the presence of a second cell attachment protein in bovine serum and present the evidence that this molecule is the bovine equivalent of vitronectin. The molecular size of this protein is in the same range as the size of the adhesive human plasma protein, vitronectin. The bovine protein also shared with human vitronectin an affinity for glass, and it could be purified by a combination of glass bead and ion exchange chromatography. The isolated bovine protein had varying proportions of an 80 and a 65 kD polypeptide. It showed immunological cross-reactivity with anti-human vitronectin and with anti-human somatomedin B. Somatomedin B is a serum peptide which has a NH2-terminal sequence identical to that of human vitronectin. The identity of the bovine protein as vitronectin was established by showing that its NH2-terminal amino acid sequence is strongly homologous with those of human vitronectin and somatomedin B. Quantitation of the adhesive activities of fibronectin and vitronectin in bovine plasma and fresh serum showed that more activity is associated with vitronectin than with fibronectin. The preponderance of vitronectin was particularly clear in fetal bovine serum intended for cell culture. In various batches, cell attachment activity attributable to vitronectin was 8-16-fold greater than that of fibronectin, making vitronectin the main adhesive protein in routine cell culture media.