Eggs of Urechis caupo are surrounded by a congruent to 0.9 micrometer thick egg envelope and, attached to that, a peripheral jelly layer about 3 micrometers thick. Before fertilization, the sperm undergoes the acrosome reaction and binds to the egg envelope. As part of a study of the induction of the acrosome reaction and sperm binding in Urechis, we have developed a method to prepare an egg envelope fraction by differential centrifugation. The isolation procedure removes much of the jelly layer, but does not alter the fine structure of the envelope. When a sperm contacts an isolated envelope, it undergoes a normal acrosome reaction and binds to the envelope's outer face. Electrophoresis of the envelope fraction on sodium dodecyl sulphate (SDS)/polyacrylamide gels revealed six major components stained by Coomassie Blue, of which four are stained by the periodic acid-Schiff reagents (PAS). To measure the degree of enrichment of the envelope fraction, envelopes were isolated from eggs that had been externally radio-iodinated; the specific activity of the envelope fraction was 17 +/- 3 times greater than that of intact eggs. The amino acid composition of the envelope fraction is dominated by Gly (19 mole %), Asx (11%), Thr (11%), Ser (8%), Ala (8%) and Glx (8%). The sugars fucose, xylose, mannose, galactose, glucose, N-acetylglucosamine and N-acetylgalactosamine were detected by gas-liquid chromatography. We also investigated whether the egg envelope changes at fertilization. No change was detected in the electrophoretic 125I pattern of externally radio-iodinated eggs, and the envelope fractions prepared from unfertilized and fertilized eggs produced the same Coomassie Blue pattern on SDS/polyacrylamide gels.