The transcriptional regulation of Escherichia coli ATCC11105 penicillin amidase (pac) gene was studied by modifying DNA sequences responsible for promoter activation by cyclic AMP receptor protein (CRP). The nucleotide sequence of the 5'-flanking region of the pac gene contains putative tandem CRP binding sites positioned at -69/-70 and at -111/-112 with respect to the transcriptional start site. Our results obtained with either point mutations or insertion or deletion mutants (each of which rotated the helix structure at the CRP binding site one-half turn) showed significant decrease of penicillin amidase (PA) activity, suggesting the CRP as a major activator. In this study, the evidence for the importance of spacing between tandem binding sites for CRP as well as for their location related to the promoter core sequence has been provided. Involvement of integration host factor (IHF) as an additional regulatory protein in the pac gene transcription regulation was also analyzed. It is shown that activation of the pac gene transcription is elevated by IHF.