A segment of DNA containing cya, the gene for adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1], has been isolated from Salmonella typhimurium. The phage lambda gt4 was used as a cloning vector and adenylate cyclase-positive hybrid phages were isolated that complemented adenylate cyclase-negative bacteria. The cloned DNA fragment encodes a polypeptide of molecular weight 81,000 that gives rise to adenylate cyclase activity. This protein represents a functional mutant of the bacterial adenylate cyclase. When the cya gene was amplified by inserting into a multicopy plasmid, the enzyme activity was overproduced 20-fold, but the cyclic AMP level increased only 60%, suggesting several probable regulatory mechanisms. Overproduction of enzymes by recombinant DNA techniques can be a useful probe of relationships in the metabolizing organism in vivo.