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Fresh human A1 erythrocytes, washed and pretreated in phosphate buffer with or without papain, were incubated at 37 degrees C with blood group-degrading enzymes from the human fecal Ruminococcus torques strain IX-70. The effects were assayed as changes in hemagglutination patterns, and blood group activities of alkali stable glycolipid extracts from the enzyme-treated cells using Dolichos biflorus anti-A1 lectin, Ulex europaeus type 1 anti-H lectin, and various monoclonal anti-A antibodies. Hemolysis was negligible (less than or equal to 1% after 6 h), and the osmotic fragility increased slightly only after papain treatment. The papain-untreated A1 erythrocytes lost D. biflorus agglutinability within minutes at room temperature with the unfractionated bacterial enzyme mixture IX-70 (42 mU 1,3-alpha-N-acetylgalactosaminidase (alpha-GalNAc'ase)/ml), but remained A active by strong agglutination with BioClone anti-A antibody even after 6 h of incubation. Thin layer chromatographic (TLC) immunostaining of extracted lipids showed hydrolysis of D. biflorus binding glycosphingolipids with more than six monosaccharides after 1 h, i.e., at a slower rate than the loss of D. biflorus agglutinability. Disappearance of these glycosphingolipids after 1 h paralleled the appearance of U. europaeus agglutinability and the strong binding of this lectin to glycolipid extracts in TLC immunoassays. A partly purified 1,3-alpha-GalNAc'ase (XI-117) (100 mU/ml) and a 1,2-alpha-fucosidase fraction (XI-50) containing alpha-GalNAc'ase (10 mU/ml) did not degrade blood group A active glycosphingolipids but completely abolished the D. biflorus agglutinability within 6 h. Papain pretreatment exposed U. europaeus receptors on the cell surface without changing the A1 hemagglutination pattern. It also facilitated a complete degradation of D. biflorus and U. europaeus reactive glycolipids with the IX-70 enzyme mixture within 6 h. The D. biflorus lectin was a good discriminator of A1/A2 subjects using erythrocyte lipid extracts but had a low affinity for the blood group A type 3 and type 4 glycosphingolipids in the TLC-overlay technique. In conclusion this study shows that (i) loss of D. biflorus A1 hemagglutination does not correlate with a loss of D. biflorus binding glycosphingolipids and (ii) loss of D. biflorus binding glycosphingolipids does not correlate with a loss of D. biflorus agglutinability. The results indicate that the serological D. biflorus agglutinability of A1 erythrocytes is not dependent on medium-sized glycosphingolipids (hexa- to dodecaglycosylceramides).