Treatment of the embryonic heart shortly after looping with 6-diazo-5-oxo-L-norleucine (DON) primarily inhibited the secretion of hyaluronate by the myocardium, an event accompanied by delayed and reduced formation of AV cushion cells (valvular and septal primordia). Sulfated glycosaminoglycan synthesis (primarily chondroitin-6-sulfate and heparan sulfate) was not significantly reduced until after cushion cell formation, an event accompanied by their altered migratory behavior. Results thus support a causal relationship between the morphogenesis of cushion tissue and extracellular glycosaminoglycans.