The lactose (lac) operon promoter is positively regulated by the catabolite gene activator-cyclic AMP complex (CAP) that binds to the DNA located 61.5 bp upstream of the transcription start site. Between the CAP binding site and the core promoter sequence is a 13-bp sequence (from -38 to -50 [the -45 region]). The possible roles of the -45 region in determining the CAP-independent level of lac expression and in the CAP activation process were studied by isolating and characterizing random multisite mutations. Only a small percentage of mutants have dramatic effects on lac promoter activity. Among the mutations that did affect expression, a 26-fold range in lac promoter activity in vivo was observed in the CAP-independent activity. The highest level of CAP-independent lac expression (13-fold the level of the wild-type lac promoter) correlated with changes in the -40 to -45 sequence and required an intact RNA polymerase alpha subunit for in vitro expression, as expected for an upstream DNA recognition element. Mutant promoters varied in their ability to be stimulated by CAP in vivo, with levels ranging from 2-fold to the wild-type level of 22-fold. Only a change of twofold in responsiveness to CAP could be attributed to direct DNA sequence effects. The -40 to -45 sequence-dependent enhancement of promoter activity and CAP stimulation of promoter activity did not act additively. The mutant promoters also displayed other characteristics, such as the activation of nascent promoter-like activities overlapping lac P1 and, in one case, replicon-dependent changes in promoter activity.