As a first step in the study of the functional consequences of molecular polymorphism at the Opaque-2 locus in maize, 1933 bp of the gene were sequenced in 21 inbred lines representative of the progenitors of cultivated varieties. High levels of polymorphism were found: 109 sites were variable in non-coding regions and 159 in protein-coding regions. Among the latter, 103 were nonsynonymous, resulting in 94 amino acid replacements in a 422-residue peptide. Moreover, 26 insertion/deletion polymorphisms were found, eight of them in coding regions. The high rate of polymorphism observed could indicate that the effective size of the ancestral population was very large, and/or that the O2 gene evolved under the influence of a high mutation rate or was subjected to some kind of balancing selection. The distribution of polymorphism within the sequence was not uniform. Silent polymorphisms were relatively frequent in exon 1 and rare in intron 5, whereas nonsynonymous polymorphisms were infrequent in the part of the sequence encoding the active domain of the transcription factor. The peptide sequence also showed a relatively higher level of resistance to amino acid replacements in this region. Sites in linkage disequilibrium were arranged mainly in three spatial patterns. This mosaic pattern can be related with an apparently large number of recombination events which might be a characteristic feature of this particular sample and/or imply that the O2 locus is a hot spot for recombination.