Developing embryos of the sea urchin, Strongylocentrotus purpuratus, incorporate [3H]palmitic acid into at least 20 proteins. The [3H]palmitic acid associated with these proteins is released by alkaline hydrolysis or by treatment with hydroxylamine but not by extensive extraction with chloroform:methanol, indicating that the fatty acids are covalently attached to protein. The finding that the fatty acid is released by hydroxylamine or beta-mercaptoethanol at neutral or even slightly acidic pH suggests that this moiety may be attached to the polypeptide via a thiol ester bond. Concanavalin A-agarose chromatography and endo-beta-N-acetylglucosaminidase H digestion revealed that 14 of the proteins containing covalently linked fatty acid also contain at least one asparagine-linked oligosaccharide chain. With one exception, all of the fatty acylated proteins are tightly associated with membranes. The rate of incorporation of [3H]palmitic acid into the proteins is developmentally regulated. Between fertilization and the onset of gastrulation (approximately 30 h), embryos exhibit a linear, 5.5-fold increase in the rate of incorporation of fatty acid into polypeptide. Incorporation increases an additional 25% during gastrulation, and then remains constant throughout subsequent development to the pluteus stage (approximately 90 h). These findings demonstrate that the fatty acylation of proteins and glycoproteins is not limited to higher organisms, since it occurs during differentiation and embryonic development of a relatively simple invertebrate.