The genomic organization of the gene encoding rat aspartyl-tRNA synthetase (AspRS), a class II aminoacyl-tRNA synthetase (aaRS), was determined. A single active gene and several pseudogenes were isolated from a rat genomic DNA library and characterized. The active DRS1 gene encoding the rat AspRS spans approximately 60 kb and is divided into 16 exons. Exons 8-16, encoding the nt-binding domain of the synthetase, are clustered in the 3'-region of the gene, whereas exons 3, 4, and 5, encoding the anticodon-binding domain are separated by large introns (up to 15 kb) containing LINE sequences. One of the pseudogenes, psi DRS1, has a nt sequence 93% identical to that of the complete cDNA sequence of rat AspRS but several stop codons interrupt the coding sequence, thus identifying psi DRS1 to an inactive processed pseudogene. Two repetitive elements from the LINE family are inserted into psi DRS1. Calculation of nt substitution rates suggests that psi DRS1 sequences arose approximately 27 Myr ago. The other pseudogene, psi DRS2, should be more ancient. Taken together, these results clearly demonstrate that the AspRS gene family is composed of only one active gene. The availability of the gene structure of AspRS could help to clarify molecular evolution of class II aaRS.