Gene manipulation techniques have been used to alter the binding site for the cyclic AMP-cyclic AMP receptor protein complex (cAMP-CRP) at the regulatory region of the Escherichia coli galactose (gal) operon. The effects of these changes on CRP-dependent stimulation of expression from the galP1 promoter in vivo have been measured, and gel binding assays have been used to measure the affinity of cAMP-CRP for the modified sites. Firstly we have deleted progressively longer sequences from upstream of the gal CRP site in order to locate the functional limit of the site. A deletion to -49, removing the first base that corresponds to the consensus sequence for a CRP binding site, is sufficient to reduce CRP binding and block CRP-dependent stimulation of P1. Secondly, we used synthetic oligonucleotides to invert the asymmetric nucleotide sequence at the gal CRP binding site or to make the sequence symmetric. Inversion of the site has little effect on CRP binding, the architecture of open complexes at P1 revealed by DNAase I footprinting, or the stimulation of transcription from P1. Making the site symmetric increases the affinity for CRP by over 50-fold and leads to increased transcription from P1, whilst hardly altering the DNAase I footprint of open complexes. Our results confirm that the strength of binding of CRP depends on the nature of the site and show that it is this that principally accounts for differences in CRP-dependent stimulation of transcription.