An investigation of di- and triglycosylceramides of hepatopancreas of a fresh-water bivalve, Hyriopsis schlegelii, gave the following results: (1) The total amount of neutral glycosphingolipid was 7.2 mg/g of the dry weight of the hepatopancreas. (2) The diglycosylceramide fraction accounted for 4.8% of the total neutral glycolipid, and consisted of Man(beta 1 leads to 2)Man(beta 1 leads to 1)Cer, Man (beta 1 leads to 4)Glc leads to Cer, Gal(beta 1 leads to 4)Glc leads to Cer, and Gal(beta 1 leads to 4)Gal(beta 1 leads to 1)Cer. The presence of so many different types of diglycosylceramides in a single tissue appears to be the exception rather than the rule. (3) The triglycosylceramide fraction amounted to 12.2% of the total neutral glycolipid and contained a mixture of Man(alpha 1 leads to 3)Man(beta 1 leads to 2)Man(beta 1 leads to 1)Cer and Man(alpha 1 leads to 3)Man(beta 1 leads to 4)Glc leads to Cer. (4) The spectrum of di- and triglycosylceramides of the hepatopancreas was more complex than that of spermatozoa of this bivalve. (5) It is an interesting finding that a series of glycolipids containing one to three mannose residues occurs in nature. (6) The ceramide moieties of the above two fractions contained the normal saturated fatty acids in the range of C14-C24, and derivatives of 4-sphingenine. The structural analysis involved (1) gas-liquid chromatography of the component sugars, fatty acids and long-chain bases; (2) methylation studies coupled with gas-liquid chromatography and mass spectrometry to locate the bonds between the hexose units; and (3) enzymatic degradation to establish the sugar sequence and the anomeric configuration of the glycosidic links.