Aspartate aminotransferase (AAT) plays a key enzymatic role in the assimilation of symbiotically fixed nitrogen in legume root nodules. In alfalfa, two distinct genetic loci encode dimeric AAT enzymes: AAT1, which predominates in roots, and AAT2, which is expressed at high levels in nodules. Three allozymes of AAT2 (AAT2a, -2b and -2c), differing in net charge, result from the expression of two alleles, AAT2A and AAT2C, at this locus. Utilizing antiserum to alfalfa AAT2, we have previously isolated from an expression library one AAT2 cDNA clone. This clone was used as a hybridization probe to screen cDNA libraries for additional AAT2 cDNAs. Four different clones were obtained, two each that encode the AAT2a and AAT2c enzyme subunits. These two sets of cDNAs encode polypeptides that differ in net charge depending upon the amino acid at position 296 (valine or glutamic acid). Within each set of alleles, the two members differ from each other by the presence or absence of a 30 bp (ten amino acid) sequence. The presence or absence of this ten amino acid sequence has no effect on the size or charge of the mature AAT2 protein because it is located within the region encoding the protein's transit peptide, which is proteolytically removed upon transport into plastids. The data suggest that a deletion event has occurred independently in two AAT2 progenitor alleles, resulting in the four allelic cDNA variants observed. The deletion of this ten amino acid sequence does not appear to impair the normal maturation of the enzyme.