The 85B protein of Mycobacterium bovis is a member of the secreted antigen 85 complex, which has been identified in a number of pathogenic mycobacteria. The 85 complex contains three components with molecular masses of 30 to 32 kDa which share the property of binding to fibronectin, a large glycoprotein present in plasma. To investigate this activity we have expressed the M. bovis 85B antigen as a recombinant protein and studied its interaction with human fibronectin. Fibronectin bound to the immobilized 85B protein in a solid-phase enzyme-linked immunosorbent assay (ELISA) and in the fluid phase in a radioimmunoassay using 125I-labelled 85B protein. In addition, fibronectin reacted with immobilized 85B in immunoblots and vice versa. Fibronectin also bound to three fragments of a cyanogen bromide digest of 85B which were subsequently identified by N-terminal sequencing. These fragments contained fibronectin-reactive peptides identified in ELISAs utilizing a set of 28 overlapping 20-mer peptides encompassing the 85B sequence. Further studies showed that the 85B protein reacted with a 32-kDa polypeptide from a limited tryptic digest of fibronectin which was identified as the collagen-binding domain. This region was confirmed as the 85B binding site by the fact that gelatin but not heparin inhibited the binding of fibronectin to 85B. These data indicate that the 85B-fibronectin interaction involves the binding of multiple regions of the 85B protein to the collagen-binding domain of fibronectin.