The extracellular calmodulin-sensitive adenylate cyclase produced by Bordetella pertussis is synthesized as a 215-kDa precursor. This polypeptide is transported to the outer membrane of the bacteria where it is proteolytically processed to a 45-kDa catalytic subunit which is released into the culture supernatant [Masure, H. R., & Storm, D. R. (1989) Biochemistry 28, 438–442]. The gene encoding this enzyme, cya A, is part of the cya Operon that also includes the genes cya B, cya D, and cya E. A comparison of the predicted amino acid sequences encoded by cya A, cya B, and cya D with the amino acid sequences encoded by hly A, hly B, and hly D genes from the hemolysin (hly) operon from Escherichia coli shows a large degree of sequence similarity [Glaser, P., Sakamoto, H., Bellalou, J., Ullmann, A., & Danchin, A. (1988) EMBO J. 7, 3997–4004]. Complementation studies have shown that HlyB and HlyD are responsible for the secretion of Hly A. (hemolysin) from E. coli. The signal sequence responsible for secretion of hemolysin has been shown to reside in its C-terminal 27 amino acids. Similarly, Cya B, Cya D, and Cya E are required for the secretion of Cya A from Bordetella pertussis. We placed the cya A gene and a truncated cya A gene that lacks the nucleotides that code for a putative C-terminal secretory signal sequence under the control of the lac promoter in the plasmid pUC-19. These plasmids were transformed into strains of E. coli which contained the hly operon. The truncated cya A gene product, lacking the putative signal sequence, was not secreted but accumulated inside the cell. However, induction of the full-length cya A gene in these strains led to cell-associated and secreted adenylate cyclase activity. In the absence of the hly operon, there was no adenylate cyclase activity secreted from E. coli containing cya A. The secreted adenylate cyclase had a molecular weight of 215K while the cell-associated activity corresponded to polypeptides of 215, 103, and 51 kDa. Partially purified preparations of the extracellular adenylate cyclase secreted by E. coli did not elevate cAMP levels in mouse neuroblastoma cells. These data indicate that the gene products of the hly operon recognized the signal sequence of cya A and facilitated the secretion of the 215-kDa precursor of B. pertussis adenylate cyclase from E. coli. © 1990, American Chemical Society. All rights reserved.