The structural gene coding for the lysine-sensitive aspartokinase II of the methylotrophic thermotolerant Bacillus sp. strain MGA3 was cloned from a genomic library by complementation of an Escherichia coli auxotrophic mutant lacking all three aspartokinase isozymes. The nucleotide sequence of the entire 2.2-kb PstI fragment was determined, and a single open reading frame coding for the aspartokinase II enzyme was found. Aspartokinase II was shown to be an α2β2 tetramer (M(r) 122,000) with the β subunit (M(r) 18,000) encoded within the α subunit (M(r) 45,000) in the same reading frame. The enzyme was purified, and the N-terminal sequences of the α and β subunits were identical with those predicted from the gene sequences. The predicted amino acid sequence was 76% identical with the sequence of the Bacillus subtilis aspartokinase II. The transcription initiation site was located approximately 350 bp upstream of the translation start site, and putative promoter regions at -10 (TATGCT) and -35 (ATGACA) were identified. A 300- nucleotide intervening sequence between the transcription initiation and translational start sites suggests a possible attenuation mechanism for the regulation of transcription of this enzyme in the presence of lysine.