Maximum expression of the adjacent but divergently transcribed araBAD operon and araC gene requires the presence of cyclic AMP (cAMP) and the cAMP receptor protein (CRP). DNase I protection studies have previously revealed a high-affinity CRP-binding site in the ara regulatory region. Deletion mutations introduced into this site resulted in reduced expression of araBAD and araC. However, other experiments have demonstrated that spacing changes in the ara regulatory region may have multiple effects due to disruption of a DNA loop. Thus, the deletions could have destroyed the CRP-binding site, the ability to form a loop, or both. In the present study, substitution mutations were introduced into the CRP site in order to avoid creating spacing changes. We found that a 3-base-pair substitution resulted in a 30% reduction in araBAD expression, whereas a 6-base-pair substitution resulted in an 80% reduction. Both of these substitution mutations reduced araC expression threefold. We conclude that CRP bound to this site regulates expression in both directions. We found that a spacing change in the CRP site does not alter araBAD expression any more than does a substitution mutation.