We have used transpositional mutagenesis of a proline auxotroph (PAO951) to isolate an ornithine utilization (oru) mutant of Pseudomonas aeruginosa (PAO951-4) that was unable to use ornithine efficiently as the sole carbon and nitrogen source. DNA sequence analysis of the inactivated locus confirmed that the transposon had inserted into a locus whose product demonstrated significant primary sequence homology to members of the AraC family of transcriptional activators. DNA mobility shift assays affirmed this potential regulatory function and indicated that the inactivated gene encodes a transcriptional regulator, which has been designated OruR. In trying to define the ornithine utilization phenotype further, a similar inactivation was engineered in the wild-type strain, PAO1. The resulting isolate (PAO1R4) was totally unable to use ornithine as the sole carbon source. Despite the intensified phenotype, this isolate failed to demonstrate significant changes in any of the catabolic or anabolic enzymes that are known to be subject to regulation by the presence of either ornithine or arginine. It did, however, show modified levels of an enzyme, ornithine acetyltransferase (OAcT), that was previously thought to have merely an anaplerotic activity. Definition of this oruR locus and its effects upon OAcT activity provide evidence that control of ornithine levels in P. aeruginosa may have a significant impact upon how the cell is able to monitor and regulate the use of arginine and glutamate as sources of either carbon or nitrogen.