The proC gene of Pseudomonas aeruginosa encodes the constitutive delta 1-pyrroline 5-carboxylate reductase (the third enzyme of proline biosynthesis) and ranks among the numerous Pseudomonas genes which are poorly transcribed in Escherichia coli. The promoters of the proC gene were located by deletion mapping. The 5' ends of the proC transcripts originating from one promoter were determined by primer extension. This promoter has a GG-N10-GC motif with a 16 bp spacing between the GC doublet and the transcription start site. Such spacing is unusually long for sigma 54-dependent promoters. In rpoN mutants of P. aeruginosa and P. putida a proC'--'lacZ fusion was expressed at wild-type levels, suggesting that sigma 54 RNA polymerase is not involved in proC transcription. The expression of another P. aeruginosa gene, anr (for anaerobic regulation of nitrate respiration and anaerobic arginine degradation), also appeared to be independent of RpoN in Pseudomonas and occurred at a very low level in E. coli. The proC and anr promoters have sequence similarities in addition to the conserved GG--N10--GC motif and may also be related to some alg (alginate) promoters of P. aeruginosa. We propose that the proC and anr promoters are activated by proteins, including perhaps an alternative sigma factor, which are present in Pseudomonas but absent from E. coli.