We have generated a series of deletions in the downstream region of the lac promoter. The promoter activities of these mutations were compared by measuring the levels of beta-galactosidase gene expression in vivo. Our results show that deletion of downstream lac promoter sequences changes the promoter strength only two- to threefold. The effects of these deletions on transcription initiation site location were studied through primer extension assay of in vivo mRNAs. We found that the transcription start sites are primarily chosen as an approximate distance from the -10 region of the lac promoter; however, starts are sometimes manifested at a GAATT(C) sequence, which is identical to the wild-type preferred start site. lac promoter P2 and a newly identified promoter, P3, are transcribed in vivo at low levels. Catabolite activator protein complexed with cyclic AMP represses P2 and P3 expression in vivo. The secondary catabolite activator protein binding site plays at most a modest role in catabolite repression in vivo.