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The three morphologically and functionally distinct jelly coat layers of Xenopus laevis eggs, J1, J2, and J3, were separated by manual dissection, solubilized with dithiothreitol, and physicochemically analyzed. The chemical composition of the three jelly layers varied from 37 to 48% protein and 63 to 52% carbohydrate. The carbohydrate consisted of hexosamines, galactose, and fucose. Some of the carbohydrate in each of the jelly layers was covalently linked to protein through O-glycosidic bonds as beta elimination of the carbohydrate moiety in the presence of alkali was observed. In agreement with a previous finding, covalently attached sulfate was localized within the innermost jelly coat layer, J1. Cellulose acetate electrophoresis at pH 8.0 resolved a total of nine macromolecular components from the three jelly coat layers differentially staining for protein and carbohydrate: J1 yielded two anodically migrating components; the middle layer J2 yielded two cathodically migrating macromolecular components; the outermost layer J3 contained five species, three anodic and two cathodic. Sodium dodecyl sulfate agarose gel electrophoresis analysis yielded nine unique species, six of which stained coincidently for protein and carbohydrate. Immunoelectrophoresis and Ouchterlony double diffusion analyses using antiserum to total jelly components resolved nine different antigenic species with cross-reactivity between one or two macromolecules in layers J1 and J3. Analytical sedimentation velocity centrifugation revealed eight distinct species all of which exhibited hypersharp schlieren patterns and whose s20,w values were highly concentration dependent. On the basis of these analyses, Xenopus laevis egg jelly layers are composed of at least 8-9 distinct macromolecular species. The majority of these macromolecules are uniquely associated with different jelly coat layers.