By utilizing a homologous transient expression system, we have demonstrated that the Opaque-2 (O2) gene product O2 confers positive trans-regulation on a 22-kD zein promoter. This trans-acting function of the O2 protein is mediated by its sequence-specific binding to a cis element (the O2 target site) present in the 22-kD zein promoter. A multimer of a 32-bp promoter fragment containing this O2 target site confers transactivation by O2. A single nucleotide substitution in the O2 target sequence not only abolishes O2 binding in vitro, but also its response to transactivation by O2 in vivo. We have also demonstrated that an amino acid domain including the contiguous basic region and the heptameric leucine repeat is essential for the trans-acting function of the O2 protein. Similar but not identical O2 target sequence motifs can be found in the promoters of zein genes of different molecular weight classes. Conversion of such a motif in the 27-kD zein promoter to an exact O2 target sequence by site-directed mutagenesis was sufficient to increase the binding affinity of the O2 protein in vitro and to confer transactivation by O2 in vivo.