Zeins, the maize (Zea mays) prolamin storage proteins, accumulate at very high levels in developing endosperm in endoplasmic reticulum membrane-bound protein bodies. Products of the multigene α-zein families and the single-gene γ-zein family are arranged in the central hydrophobic core and the cross-linked protein body periphery, respectively, but little is known of the specific roles of family members in protein body formation. Here, we used RNA interference suppression of different zein subclasses to abolish vitreous endosperm formation through a variety of effects on protein body density, size, and morphology. We showed that the 27-kilodalton (kD) γ-zein controls protein body initiation but is not involved in protein body filling. Conversely, other γ-zein family members function more in protein body expansion and not in protein body initiation. Reduction in both 19- and 22-kD α-zein subfamilies severely restricted protein body expansion but did not induce morphological abnormalities, which result from reduction of only the 22-kD α-zein class. Concomitant reduction of all zein classes resulted in severe reduction in protein body number but normal protein body size and morphology.