The Opaque-2 gene (O2) in maize encodes a transcriptional activator that controls the expression of various genes during kernel development, particularly some of the most abundant endosperm storage protein genes. Compared to its wild relative teosinte, maize has bigger and heavier kernels, with an increased proportion of starch and an altered distribution of the various storage protein categories. The molecular evolution of the O2 gene was investigated in connection with its possible involvement in the domestication process. Most of the coding sequence and parts of introns, 5'UTR, and 3' noncoding regions were sequenced in a set of cultivated and teosinte accessions. One hundred six polymorphic sites (5.4%) and 72 insertions/deletions, located mostly in noncoding regions, were found. Molecular diversity was quite high (pi = 0.0138, theta = 0.0167) compared to that of other transcription factors in maize. The synonymous and nonsynonymous diversity patterns along the coding sequence suggested that different regions are submitted to different functional constraints. Such an evolution would probably be favored by the observed rapid decay of linkage disequilibrium with distance. Cultivated accessions retained about 70% of the diversity observed in teosintes. Purifying selection was detected in both maize and teosintes. No conclusive evidence was obtained for a role of the O2 gene in the domestication process.