Results from a random mutagenesis procedure on the PcaU binding site from Acinetobacter baylyi followed by in vivo and in vitro screening are presented. PcaU is an IclR-type transcriptional regulator from the soil bacterium A. baylyi and is required for the regulated expression of enzymes for protocatechuate and quinate degradation encoded by the pca-qui operon. It binds to a 45 bp area located in the pcaU-pcaI intergenic region which consists of three perfect 10 bp sequence repeats forming one palindrome (R1, R2) and an additional direct sequence repeat (R3). In vivo selection for pca-qui gene expression revealed that mutations within the three sequence motifs are tolerated to different extents. The functional requirement for conserved nucleotides was greatest in the external half of the palindrome (R1). Four positions within and directly adjacent to this 10 bp sequence never acquired a mutation, and are therefore considered to be the most important for transcriptional regulation by PcaU. Transcriptional output is affected in different ways; for some of these changes there is a correlation with a reduction in the affinity of PcaU for these sites. Two of these positions were also preserved when in vitro screening was performed for PcaU binding alone. Additional conserved residues are detected by the in vitro approach, indicating that the regions of the PcaU binding site involved in binding differ, at least in part, from those required for functional gene expression.