In the maize endosperm, the expression of the subfamily 4 (SF4) of the zein genes is under the transcriptional control of the Opaque2 (O2) basic leucine zipper transcriptional activator, which binds to the O2-box 5'-TCCACGTAGA-3'. Southern experiments showed that the O2-box core sequence ACGT is heavily methylated in sporophytic tissues but becomes hypomethylated in the endosperm. DNA analyses of two inbred lines and their reciprocal crosses indicate that the hypomethylation state is established on the maternal complements of the endosperm. Electrophoretic mobility shift assay (EMSA) and southwestern experiments with endosperm protein extracts and recombinant O2 using oligonucleotides with methylated and unmethylated cytosines in the O2-box indicate an inhibitory effect of modified sequences on O2-binding activity. These results suggest that DNA methylation modulates O2 activity in vivo and shed light on molecular mechanisms involved in the parent-dependent zein gene expression in maize endosperm.