To investigate the level and pattern of DNA polymorphism in the noncoding regulatory region in the plant nuclear genome, 2.4 kb of nucleotide sequence of the 5' upstream region of ADH: was determined for 14 ecotypes of Arabidopsis thaliana and five accessions of Arabis gemmifera. Using this data set and previously determined ADH: sequence data, DNA variation was analyzed in a 4.4-kb region of the locus. Two divergent sequence types detected in the transcriptional unit of ADH: were not present in the 5' region of the ADH: gene in A. thaliana. Nucleotide diversity of the entire 5' region was estimated to be 0.0040, which is lower than that in the transcriptional unit. The level of variation was not uniform. There were peaks of variations in a approximately 400-bp region where cis-regulatory elements for ADH: expression were clustered and in exon 4. In interspecific comparison with A. gemmifera, lower divergence was observed in the 5' flanking region than in the exons. High peaks of divergence in the 400-bp regulatory region and exon 4 were also detected, although there were many other peaks. These results indicate that regions of functional importance have a high level of polymorphism and divergence in the ADH: locus of these genera. The possibility of balancing selection in the ADH: gene of these plants is discussed.