The hyaline layer (HL) is an apically located extracellular matrix (ECM) which surrounds the sea urchin embryo from the time of fertilization until metamorphosis occurs. While gelatin-cleavage activities were absent from freshly prepared hyaline layers, a dynamic pattern of activities developed in layers incubated at 15 or 37 degrees C in Millipore-filtered sea water (MFSW). Cleavage activities at 90, 55, 41, and 32 kDa were evident following incubation at either temperature. The activation pathway leading to the appearance of these species was examined to determine the minimum salt conditions required for processing and to establish precursor-product relationships. In both qualitative and quantitative assays, the purified 55 kDa gelatinase activity was inhibited by 1,10-phenanthroline (a zinc-specific chelator) and ethylenebis (oxyethylenenitrilo) tetraacetic acid (EGTA). Calcium reconstituted the activity of the EGTA-inhibited enzyme with an apparent dissociation constant (calcium) of 1.2 mM. Developmental substrate gel analysis was performed using various stage embryos. The 55 and 32 kDa species comigrated with gelatin-cleavage activities present in sea urchin embryos. Collectively, the results reported here document a zymogen activation pathway which generates a 55 kDa, gelatin-cleaving activity within the extraembryonic HL. This species displayed characteristics of the matrix metalloproteinase class of ECM modifying enzymes.