Specific and high titer antisera against 4-O-methylglucuronic acid-containing glycosphingolipid (Lipid IV) from spermatozoa of the fresh-water bivalve, Hyriopsis schlegelii, were raised in rabbits. The antisera were found to agglutinate spermatozoa of three fresh-water bivalves, H. schlegelii, Anodonta woodiana, and Cristaria plicata (Palaeoheterodonta), but they did not agglutinate those of Corbicula sandai (Heterodonta). The specificity of the agglutination was examined by an inhibition test using various carbohydrates, from which it was concluded that an antigenic determinant is GlcA4Me-Fuc. Immunohistochemical studies indicated that Lipid IV exists on the cell surface of the spermatozoa.