Escherichia coli purB encodes adenylosuccinate lyase (ASL), the enzyme that catalyzes step 8 in the pathway for de novo synthesis of IMP and also the final reaction in the two-step sequence from IMP to AMP. Gene purB was cloned and found to encode an ASL protein of 435 amino acids having a calculated molecular weight of 49,225. E. coli ASL is homologous to the corresponding enzymes from Bacillus subtilis and chickens and also to fumarase from B. subtilis. Gene phoP is 232 bp downstream of purB. Gene purB is regulated threefold by the purine pool and purR. Transcriptional regulation of purB involves binding of the purine repressor to the 16-bp conserved pur regulon operator. The purB operator is 224 bp downstream of the transcription start site and overlaps codons 62 to 67 in the protein-coding sequence.