Opaque or nonvitreous phenotypes relate to the seed architecture of maize (Zea mays) and are linked to loci that control the accumulation and proper deposition of storage proteins, called zeins, into specialized organelles in the endosperm, called protein bodies. However, in the absence of null mutants of each type of zein (i.e. alpha, beta, gamma, and delta), the molecular contribution of these proteins to seed architecture remains unclear. Here, a double null mutant for the delta-zeins, the 22-kD alpha-zein, the beta-zein, and the gamma-zein RNA interference (RNAi; designated as z1CRNAi, betaRNAi, and gammaRNAi, respectively) and their combinations have been examined. While the delta-zein double null mutant had negligible effects on protein body formation, the betaRNAi and gammaRNAi alone only cause slight changes. Substantial loss of the 22-kD alpha-zeins by z1CRNAi resulted in protein body budding structures, indicating that a sufficient amount of the 22-kD zeins is necessary for maintenance of a normal protein body shape. Among different mutant combinations, only the combined betaRNAi and gammaRNAi resulted in drastic morphological changes, while other combinations did not. Overexpression of alpha-kafirins, the homologues of the maize 22-kD alpha-zeins in sorghum (Sorghum bicolor), in the beta/gammaRNAi mutant failed to offset the morphological alterations, indicating that beta- and gamma-zeins have redundant and unique functions in the stabilization of protein bodies. Indeed, opacity of the beta/gammaRNAi mutant was caused by incomplete embedding of the starch granules rather than by reducing the vitreous zone.