After fertilization, sea urchin embryos surround themselves with an extracellular matrix, or hyaline layer, to which cells adhere during early development. Hyalin, the major protein component of the hyaline layer has been isolated and partially characterized in several laboratories. Although other proteins are present in the hyaline layer, little is known about their origin, distribution, or functions. The present report characterizes a set of hyaline layer proteins that are secreted after fertilization from a class of vesicles that are distinct from cortical granules. The group of proteins in these vesicles were identified by a monoclonal antibody (8d11) which recognizes a carbohydrate epitope common to each of these molecules. 8d11 polypeptides range in molecular weight from 105 to 225 kDa. Oogonia and oocytes in early stages of vitellogenesis do not express the antigen. The proteins are first observed by immunofluorescence during oogenesis as a peripheral band in mid-vitellogenic oocytes. Following germinal vesicle breakdown 8d11 moves to be distributed evenly throughout the cytoplasm. The proteins are transported to the egg surface by a cytochalasin-sensitive mechanism after fertilization, and secreted predominately within the first 30 min of development. 8d11 proteins are depleted in areas of cell contact during early embryogenesis, and become concentrated on the apical surface of ectoderm cells where they are assembled into high-molecular-weight aggregates. Three of the molecules in this group may be proteins previously described as "apical lamina" proteins. These observations provide evidence of a third pathway (cortical granules and basal lamina granules being the other two) for synthesis, storage, and exocytosis of matrix proteins that are release after fertilization.