Protein was isolated from the ring-shaped acrosomal granules of Urechis sperm which bound tightly to Urechis egg surface coats and also initiated development. The acrosomal protein preparation migrated as a single major band in acetic acid-urea PAGE, had a lysine + arginine content of ∼50%, and lacked carbohydrate. The molecular mass of the acrosomal protein was 25,000–30,000 Da in cetyltrimethylammonium bromide PAGE. Acrosomal rings of acrosomereacted sperm were selectively labeled with fluorescamine and the fluorescence persisted throughout the isolation procedure and was observed in the major band on gels. Although the acrosomal protein and Urechis sperm protamine had similar amino acid contents and migrated similarly in acetic acid-urea gels, acrosomal protein differed from protamine by its relative insolubility in hot 5% trichloroacetic acid and cold 0.25 N H 2SO 4, by its migration in cetyl- and tetratrimethylammonium bromide PAGE and in a major spot on its peptide map following thermolysin digestion. Agglutination of eggs by Urechis acrosomal protein was not species-specific and included various echinoderm eggs and algal zygotes as well as Urechis eggs. Both the acrosomal protein preparation and protein extracted from the major band of gels initiated development of Urechis eggs, causing rounding out, elevation of the surface coat, germinal vesicle breakdown, polar body formation, and establishment of a polyspermy block. Polylysine and calf thymus histones were equally effective in activating Urechis eggs, but Urechis sperm protamine was less effective, and salmon sperm protamine, although highly basic, activated only a small percentage of eggs. Urechis acrosomal protein also induced partial elevation of sea urchin egg fertilization envelopes, similar to the response of sea urchin eggs to Urechis sperm. Sea urchin eggs were not activated by polylysine.