A second adenylate cyclase (cya2) gene was isolated from a Rhizobium meliloti F34 gene bank. Complemented E. coli delta cya mutants were capable of utilizing a number of, but not all, carbon sources known to be regulated by cAMP. DNA hybridization studies showed cya2 to be unique to R. meliloti strains. The cya2 nucleotide sequence was determined and found to encode a protein of 363 amino acids. Residues were identified within the C-terminal domain which are conserved in both eukaryotic adenylate and guanylate cyclases, including a putative ATP binding site. Similar residues were also found in the prokaryotic R. meliloti Cya1 protein. A R. meliloti cya1/cya2 double mutant was constructed and characterized; however, cAMP production was still observed in this strain indicating the presence of a third cya gene.