We studied the molecular binding specificity of two rabbit polyclonal sera generated against phenolic glycolipid antigens namely PheG1 B and PheG1 B-3 from Mycobacterium bovis BCG. PheG1 B is the well-known mycoside B (2-O-Me-alpha-L-Rhap 1----aglycone), while PheG1 B-3 is a recently found glycolipid (alpha-L-Rhap-(1----3)-2-O-Me-alpha-L-Rhap 1----aglycone). The interaction specificity was mainly explained in terms of the cavity volume of the antibodies paratope. The anti-PheG1 B antibodies paratope fits the 2-O-Me-alpha-L-Rhap ligand, while that of anti-PheG1 B-3 binds the disaccharide moiety of PheG1 B-3, and, with a higher affinity, the monosaccharidic unit localized at the non-reducing end. The B-3 antigen affinity is higher than that of antigen B for their homologous antibodies. This can be explained by the fact that the antibodies against phenolic glycolipid B-3 bind optimally to two sequential glycosyl residues suggesting the presence of two subsites. The immunoglobulin subsite with the major affinity binds the monosaccharidic unit localized at the non-reducing end.