Comparison of the cya loci (cya codes for adenylyl cyclase (AC)) from a variety of phylogenetically divergent facultative anaerobic Gram-negative bacteria reveals conserved sequence features. The entire locus structure in enterobacteria is preserved, including two major promoters (a conserved cya strong promoter, P2, and a divergent promoter for a heme biosynthetic operon, hemCD) present in the upstream region of the cya gene. The region between hemC and cya is much longer in Proteus mirabilis than in other enterobacteria, and lacks the P1 upstream cya promoter. In Aeromonas hydrophila the cya promoter (the strong P2 promoter in E coli) is preserved, including a putative GATC methylation site situated immediately downstream from the -10 box. Each cya frame analyzed uses TTG as the translation start codon and is preceded by an unusual ribosome binding site. This suggests that a lower translation efficiency of the cya transcript could be the result of some selection pressure. This has been substantiated by in vitro mutagenesis and by selection of up mutations which all map at the cya ribosome binding site. In enterobacteria the cyaY frame is the only conserved reading frame downstream of cya, with the orientation opposite to that of cya. This organization is not preserved in Aeromonas. Experiments involving fusions with the lacZ gene demonstrated that cyaY is expressed. Finally, comparison of the different polypeptide sequences of ACs permits discussion of important features of the catalytic and regulatory centers of the protein.