The binding motifs of the immunodominant antigen (Ag) alpha-Ag (Ag 85 complex B) of Mycobacterium kansasii for human fibronectin were examined using digested fragments. We defined two fibronectin-binding epitopes on 27 amino acids from 84 to 110 and on 20 amino acids from 211 to 230, The epitopes were almost conserved in the closely related Ag 85 complex of other mycobacteria species. Inhibition of fibronectin binding to intact alpha-Ag molecules was observed with peptide-(84-110), but not with peptide-(211-230). Peptide (84-110) could also inhibit fibronectin binding to all components of the Ag 85 complex of Bacillus Calmette-Guerin (Ag 85A, Ag 85B, and Ag 85C). Further study with synthetic peptides defined 11 residues from 98 to 108 as the minimum motif. Six residues ((98)FEWYYQ(103)) were critical for interacting with fibronectin. The motif revealed no homology to other known prokaryotic and eukaryotic fibronectin-binding proteins. The defined motif of alpha-Ag is novel and unique for mycobacteria.