The transcriptional regulation of NADP-malate dehydrogenase (NADP-MDH) was analyzed in Arabidopsis ecotypes and other Brassicaceae. The amount of transcript increased twofold after transfer into low temperature (12 degrees C) or high light (750 microE) in all species. Analysis of the genomic DNA reveals that the NADP-MDH gene (At5g58330 in A. thaliana) in Brassicaceae is located between two other genes (At5g58320 and At5g58340 in Arabidopsis), both encoded on the opposite DNA strand. No promoter elements were identified in 5' direction of the NADP-MDH gene, and the expression of NADP-MDH was not affected in knock-out plants carrying a DNA insert in the 5' region. A yeast-one hybrid approach yielded only three DNA-binding proteins for the 500-bp fragment located upstream of the ATG sequence, but 34 proteins for its coding region. However, in Chlamydomonas and in some Poaceae, which do not possess any genes within the 1200 bp upstream region, typical promoter elements were identified. Alignments of genomic DNA reveal that, in contrast to Poaceae, the introns are highly conserved within Brassicaceae. We conclude that in Brassicaceae the majority of regulatory elements are located within the coding region. The NADP-MDH gene of both families evolved from a common precursor, similar to the gene in Chlamydomonas. Changes in the selection pressure allowed the insertion of At5g58340 into the promoter region of a common ancestor. When the demand for transcriptional regulation increased, At5g58340 disappeared in Poaceae, and a promoter developed in the 5' region. In contrast, Brassicaceae maintained At5g58340 and shifted all regulatory elements into the coding region of NADP-MDH.