Prolamin-containing protein bodies in maize endosperm are composed of four different polypeptides, the -, ß-, -, and -zeins. The spatial organization of zeins within the protein body, as well as interactions between them, suggests that the localized synthesis of -zeins could initiate and target protein body formation at specific regions of the rough endoplasmic reticulum. To investigate this possibility, we analyzed the distribution of mRNAs encoding the 22-kD -zein and the 27-kD -zein proteins on cisternal and protein body rough endoplasmic reticulum membranes. In situ hybridization revealed similar frequencies of the mRNAs in both regions of the endoplasmic reticulum, indicating that the transcripts are distributed more or less randomly. This finding implies that zein protein interactions determine protein body assembly. To address this question, we expressed cDNAs encoding -, ß-, -, and -zeins in the yeast two-hybrid system. We found strong interactions among the 50-, 27-, and 16-kD -zeins and the 15-kD ß-zein, consistent with their colocalization in developing protein bodies. Interactions between the 19- and 22-kD -zeins were relatively weak, although each of them interacted strongly with the 10-kD -zein. Strong interactions were detected between the - and -zeins and the 16-kD -zein and the 15-kD ß-zein; however, the 50- and 27-kD -zeins did not interact with the - and -zein proteins. We identified domains within the 22-kD -zein that bound preferentially the - and -zeins and the ß- and -zeins. Affinities between zeins generally were consistent with results from immunolocalization experiments, suggesting an important role for the 16-kD -zein and the 15-kD ß-zein in the binding and assembly of -zeins within the protein body.