Escherichia coli cells that are deficient in the cAMP receptor protein (CRP) overproduce cAMP. We and others have previously found that transcription of the adenylate cyclase gene (cya) is negatively regulated by the CRP-cAMP complex. Here, we have investigated the contribution of this transcriptional regulation to the control of cAMP levels. Several variants of the cya gene have been constructed and characterized with respect to their expression and their ability to produce cAMP. Overproduction of cAMP in a crp- background was reduced from 200-fold to 50-fold when transcriptional regulation by CRP-cAMP was eliminated by replacing the cya promoter with the constitutive bla promoter. When the C-terminal 48 amino acids of adenylate cyclase were deleted without changing the promoter, the degree of overproduction of cAMP was reduced to 4-fold. Finally, the increase in cAMP level observed in crp- cells was almost completely abolished when the truncated cyclase was expressed from the bla promoter. We conclude that transcriptional regulation of cya does indeed play a role in the down-regulation of cAMP production by CRP, although the major regulation is exerted at the posttranscriptional level. The C-terminal region comprising the last 48 amino acids of cyclase is responsible for the posttranscriptional regulation. A simple new method for the determination of cAMP is also described.