Previous in vivo studies using drugs that inhibit the N-glycosylation of proteins have demonstrated that newly synthesized N-linked glycoproteins are required for gastrulation in embryos of two species of sea urchins, Strongylocentrotus purpuratus and Arbacia punctulata. To understand the biochemical events regulating glycoprotein synthesis during gastrulation in S. purpuratus embryos, we examined the in vitro activities of enzymes catalyzing several of the early steps in N-linked glycoprotein synthesis. The activities of glycosyl transferases responsible for production of N,N-diacetylchitobiosylpyrophosphoryldolichol and glucosylphosphoryldolichol, two intermediates in the formation of oligosaccharylpyrophosphoryldolichol (the carbohydrate donor for N-glycosylation), were low but detectable in membranes from eggs. After fertilization these activities remained constant or increased slowly up to the blastula stage and thereafter increased rapidly at gastrulation. In agreement with these in vitro findings, in vivo labeling experiments revealed that the rate of incorporation of [3H]Man into oligosaccharylpyrophosphoryldolichol and into protein increased three- to fourfold prior to gastrulation and then slightly more at the prism stage. In contrast, in vitro activity of mannosylphosphoryldolichol synthase, another enzyme in the pathway of N-linked glycosylation, was maximal in membranes from egg and embryos in the early stages of development and declined prior to gastrulation. Furthermore, the level of this activity was at least 100-fold greater than that for enzymes involved in the formation of the chitobiosyl and glucosyl lipids. With the exception of mannosylphosphoryldolichol synthase activity, these data indicate that there is a general activation of the glycosylation apparatus before gastrulation in sea urchin embryos. Possible explanations for the decrease in mannosylphosphoryldolichol synthase activity are discussed.