A truncated derivative of the XylR protein, which is able to constitutively activate the sigma 54-dependent Pu promoter of the TOL (toluene biodegradation) plasmid of Pseudomonas putida, has been purified to homogeneity and its various activities have been separately examined, in vitro. The truncated regulator XylR delta A was deleted of the signal reception N-terminal module present in wild-type XylR, but retained its central activation domain and the DNA binding segment, located at its C terminus. XylR delta A bound to the region -120 to -190 bp upstream of the transcription initiation site of the Pu promoter, where previous analyses have located the XylR target site. XylR delta A showed an intrinsic ATPase activity that was strongly stimulated by DNA containing the native upstream activation sequences of Pu. Both ATPase activity and ATP binding were abolished in mutant G268N in which the Walker A domain of the central module was altered. Mutant R453H lacked ATPase activity but retained the nucleotide-binding ability of the parental protein. XylR delta A was able to activate transcription in vitro with sigma 54-RNA polymerase alone, although its activity was enhanced up to 20-fold in the presence of the integration host factor protein. The requirements for activation of the Pu promoter in vitro are consistent with the view that DNA-facilitated oligomerization of the regulator for an enhanced ATPase activity is the critical event that precedes transcription initiation at sigma 54-dependent promoters. Furthermore, additional co-regulation elements seem to adjust promoter activity in vivo to the physiological status of the cells.