The cytosolic and mitochondrial isozymes of aspartate aminotransferase (AspAT) function in the C4 photosynthetic cycle in NAD-malic enzyme-type C4 plants and are expressed at high levels in mesophyll cells and bundle sheath cells, respectively. We constructed a genomic library from Panicum miliaceum, a NAD-malic enzyme-type C4 plant, and cloned the genes for these isozymes. The sequence of the cloned gene for cytosolic AspAT spans 7800 bp and consists of 12 exons. The sequence of the cloned gene for mitochondrial AspAT spans 9000 bp and consists of 10 exons. The results of primer-extension analysis suggest that transcription may be initiated from multiple adjacent sites. Both genes have significant GC-rich regions around the site of initiation of transcription, and these regions showed no CpG suppression. The 5'- flanking regions of both genes include several short sequences similar to the regulatory elements found in other genes for components of the photosynthetic machinery. In particular, the cytosolic AspAT gene contains sequences similar to nuclear protein-binding sites in other mesophyll-expressed C4 photosynthetic genes and the mitochondrial AspAT gene contains elements for light-sensitive and constitutive expression of a bundle sheath-expressed gene. The results of Southern analysis indicated that there are at least two genes that encode each isozyme in the genome of P. miliaceum. A comparison of intron-insertion positions between AspAT genes of plants and animals revealed that several introns are located at identical positions. On the basis of a phylogenetic tree among AspATs and tyrosine aminotransferase, we have shown that the introns of aminotransferase genes antedate the divergence of eubacteria, archaebacteria, and eukaryotes.