The occ and noc regions in octopine and nopaline Ti plasmids, respectively, are responsible for the catabolism of octopine and nopaline in Agrobacterium. The functions are activated in the presence of the opines by OccR and NocR, two related regulatory proteins, and the promoters contain common sequence motifs. We have investigated heterologous interactions between the regulators and the promoters. Previous experiments using all possible heterologous combinations of opines, regulators, and promoters in vivo had demonstrated that only the combination of nopaline, NocR, and the occ promoter led to limited promoter activation. We now show that OccR and NocR bind to the heterologous promoters in vitro and in vivo. The weak or non-existent promoter activation actually observed could be explained by the assumption that OccR and NocR use different activation mechanisms; we investigated protein-induced DNA bending because of reports that the two regulators differ in this respect. Analysis with a bending vector showed that both OccR and NocR induced a DNA bend that is relaxed in the presence of the respective opine. The data suggest that subtle differences in regulator/promoter interactions are responsible for the inactivity of the heterologous combinations. Investigations with a chimeric NocR/OccR protein indicated that it induced a DNA bend in both promoters. No opine-induced relaxation was detectable with the hybrid, and the inducible promoter was not activated. These findings suggest that bend relaxation may be an integral part of promoter activation.