The nahR gene of plasmid NAH7 of Pseudomonas putida encodes a 36-kilodalton polypeptide which activates transcription of the nah and sal operons in response to the inducer salicylate. A gel mobility shift assay was used to identify a DNA-binding activity which was present only in extracts from either P. putida or Escherichia coli containing a functional nahR gene. The binding activity was highly specific for DNA containing the nah or sal promoters, but the apparent affinity for the promoters was not altered by the presence of salicylate. DNase I protection experiments with a partially purified NahR protein preparation showed that NahR protects both nah and sal promoter sequences between -82 and -47. The location and amount of protection were not dramatically altered by the presence of salicylate. In vitro mutagenesis was used to make mutations in the protected region of the sal promoter. Analysis of the mutants showed that binding of NahR is required for transcription activation and identified two nucleotides in the protected region that are essential for binding and activation by NahR.