S1 nuclease was used to generate a series of deletions which extend into the CAP-cAMP binding site from upstream of the Escherichia coli lactose operon promoter (lacP). Deletion and insertion mutations were also created which changed the spacing region between the CAP-cAMP binding site and the lacP -35 region. The promoter activities of these mutations were compared by measuring the levels of beta-galactosidase gene expression in vivo. The results show that sequence information prior to 74 base pairs (-74) upstream from the transcription start site (designated as +1) is not necessary for the full activation of the lac promoter by the CAP-cAMP complex. However, the deletion which extends to the -71 position retains only one third of the promoter activity in the presence of the CAP-cAMP complex. Removal of one symmetrical element from the two fold symmetry in the CAP-cAMP binding site abolished the CAP-cAMP stimulation of the lac promoter. Spacer mutations which increase by one base pair or decrease by two base pairs the length of the spacing region between the CAP-cAMP binding site and the lacP -35 region drastically reduced the CAP-cAMP stimulation of the lac promoter. This suggests that the distance between the lac promoter transcription start site and CAP-cAMP binding site is crucial for the function of the lac promoter, despite the fact that this distance varies in other E. coli promoters positively regulated by CAP-cAMP. A deletion which extends to the -59 position results in a two fold enhanced expression of lac in the absence of CAP-cAMP. This is consistent with the existance of a competitive RNA polymerase binding site in this region which would normally act to inhibit RNA polymerase binding.