The Pseudomonas putida meta-cleavage operon encodes the enzymes for the catabolism of alkylbenzoates. Activation of meta-operon transcription is mediated by the XylS protein which, upon activation by effectors, binds two sites between -70 and -35 with respect to the main transcription initiation point at the Pm promoter. Two naturally occurring regulators, XylS and XylS1, that differ by only five amino acids, have been analyzed with regard to potential interactions of these positive regulators with the C-terminal domain of the alpha subunit of RNA polymerase (alpha-CTD). For these studies we expressed a derivative of alpha deprived of the entire C-terminal domain (alpha-Delta235) and found that expression from Pm with XylS or XylS1 was significantly decreased. To discern whether alpha-CTD activation depended on interactions with DNA and/or XylS proteins we tested a large collection of alanine substitutions within alpha-CTD. Most substitutions that had an effect on XylS and XylS1-dependent transcription were located in or adjacent to helix 1 and 4, which are known to be involved in alpha-CTD interactions with DNA. Two alanine substitutions in helix 3 (residues 287 and 291) identified a putative region of alpha-CTD/XylS regulator interactions.