During growth of the ovarian follicle, the mammalian oocyte becomes surrounded by an acellular coat called the zona pellucida. Whether the zona pellucida originates from the oocyte, surrounding follicle cells, or both has remained an unresolved issue. In experiments described here, denuded and follicle-enclosed mouse oocytes at various stages of growth were isolated and cultured in vitro in the presence of either [(35)S]methionine or [(3)H]fucose in order to determine the site of synthesis of the three, recently identified, zona pellucida proteins, ZP1, ZP2, and ZP3 [Bleil, J. D. & Wassarman, P. M. (1980) Dev. Biol., in press]. Approximately 1.5% of the [(35)S]methionine, and as much as 45% of the [(3)H]fucose, that was incorporated into trichloroacetic acid-insoluble material by denuded or follicle-enclosed oocytes during a 12-hr culture period was found associated with zonae pellucidae removed from the cultured oocytes. Incorporation of [(35)S]methionine into zona pellucida proteins was depressed to less than 1/50th when denuded oocytes were cultured in the presence of puromycin, and secretion of zona pellucida proteins by denuded oocytes was demonstrated by pulse-chase experiments. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis of [(35)S]methionine- and [(3)H]fucose-labeled proteins present in oocytes, zonae pellucidae, and follicle cells revealed that denuded oocytes synthesize and secrete zona pellucida proteins, whereas no evidence was obtained to suggest that follicle cells synthesize these proteins. Denuded oocytes, ranging in diameter from 48 to 68 mum, incorporated both [(35)S]methionine and [(3)H]fucose into zona pellucida proteins during culture in vitro, whereas zonae pellucidae removed from fully-grown oocytes (85 mum) were not radiolabeled to a significant extent. After culture of denuded or follicle-enclosed oocytes for 12 hr, more than 95% of the [(3)H]fucose incorporated into oocyte proteins was found in ZP1, ZP2, and ZP3, indicating that the zona pellucida proteins are the major class of proteins glycosylated during oocyte growth. These results provide biochemical evidence supporting the idea that the zona pellucida originates from the mammalian oocyte itself, rather than from the surrounding follicle cells.