The sea urchin embryo at the blastula stage hatches from its protective fertilization envelope which is degraded by a secreted protease, the hatching enzyme. We have previously purified the hatching enzyme from Paracentrotus lividus (Lepage and Gache (1989). J. Biol. Chem. 264, 4787-4793), cloned its cDNA, and analyzed the temporal expression of its gene (Lepage and Gache (1990). EMBO J. 9, 3003-3012). We study here the temporal and spatial expression of the hatching enzyme gene in whole embryos by immunolabeling with an affinity-purified polyclonal antibody and by in situ hybridization using nonradioactive RNA probes. The timing of expression is consistent with our data on the activation of the gene, the mRNA accumulation in the blastula, and the role of the enzyme. Immunolabeling was observed only in blastula stage embryos; neither before the 128-cell stage nor after hatching. The distribution of the enzyme varies with time from a diffuse labeling around the nucleus to a punctate localization between the nucleus and the apical face of the blastomeres, and finally at the time of hatching, to a submembranous apical location. Not all the cells of an embryo are labeled. The presence of the hatching enzyme is restricted to a sharply delimited continuous territory spanning about two-thirds of the blastula. The orientation of this territory has been determined with respect to the animal-vegetal axis of the embryo using as a landmark the subequatorial pigmented band of the P. lividus species. The synthesis of the hatching enzyme only takes place in the animal-most two-thirds of the blastula. By in situ hybridization, the mRNA coding for the hatching enzyme is only detected in early blastulas, in a limited area having the same size and shape as the territory in which the protein is found. Thus the hatching enzyme gene is likely to be spatially controlled at the transcriptional level: its expression is restricted to a region of the blastula that corresponds roughly to the presumptive ectoderm territory. To date, the hatching enzyme gene products constitute the earliest molecular markers of the sea urchin embryo spatial organization along the primordial egg axis.