Opaque-2 (O2) encodes a transcriptional activator of the basic domain-leucine zipper (bZIP) class, which controls the expression level in maize endosperm of the 22kD alpha-zeins and a number of non-storage proteins. The interaction of the O2 protein at three clustered binding sites on an isolated 22 kD zein gene promoter has been investigated. O2 is shown to transactivate transcription from these sites in tobacco mesophyll protoplasts as well as in maize endosperm cells transformed by particle bombardment. The binding sites have been mutated by base exchanges, singly or in different combinations, to determine their contribution to transactivation in vivo in both the leaf protoplast and the maize endosperm system. The effect of these mutations on binding of O2 in vitro was determined by electrophoretic mobility shift assays (EMSA), using O2 protein expressed in E. coli. Two of the sites seemed to be equally effective in responding to Opaque-2 in vivo in both cell types, although one of them does not contain an ACGT core sequence, and has a lower affinity for O2 in vitro than the ACGT-containing binding site. A third site, which has the lowest affinity of all three, confers no detectable O2-dependent promoter activation alone, but significantly increases activation in combination with either one of the other sites. Hence, weaker O2 binding sites can still mediate major O2-dependent effects when present in target promoters in vivo.