A DNA segment that promotes gene expression in Pseudomonas putida was identified in pTN8, a mutant plasmid of an RP4-TOL recombinant. A promoter on the segment was cloned with a promoter-probe vector containing the xylE gene of the TOL plasmid. The xylE gene was expressed under the control of the promoter, and the gene product catechol 2,3-dioxygenase was constitutively synthesized. As analyzed by an S1 nuclease protection assay, the amount of mRNA produced in P. putida was more than that in Escherichia coli. Fine S1 nuclease mapping and reverse transcriptase mapping revealed three tandem transcription start sites in both P. putida and E. coli. The nucleotide sequence preceding the transcription start sites was determined; a part of this sequence contained a sequence homologous to E. coli promoter sequences. A tentative consensus sequence for P. putida constitutive promoters is proposed.