The 2-N-acetyltransferase [AAC(2)-Ia] in Providencia stuartii has a dual function where it is involved in the acetylation of peptidoglycan and certain aminoglycosides. A search for negative regulators of the aac(2)-Ia gene has resulted in the identification of aarC. A missense allele (aarC1) resulted in an 8.9-fold increase in ␤-galactosidase accumulation from an aac(2)-lacZ transcriptional fusion. Northern blot analysis demonstrated an increase in aac(2)-Ia mRNA accumulation that was specific to cells at high density. In addition, the aarC1 allele also resulted in a substantial increase in the expression of aarP, a transcriptional activator of the aac(2)-Ia gene. The wild-type aarC gene was isolated by complementation and encodes a predicted protein of 365 amino acids with a molecular mass of 39,815 Da. The predicted AarC protein exhibited 88% amino acid homology to the previously identified GcpE protein of Escherichia coli and 86% homology to a gene product from Haemophilus influenzae. The E. coli gcpE gene was able to functionally complement the aarC1 allele in P. stuartii. The aarC1 allele was identified as a T to G transversion that resulted in a valine to glycine substitution at position 136 in the AarC protein.The aarC gene appears to be essential for cell viability as construction of a disrupted copy (aarC::lacZ) was possible only in cells that carried an episomal copy of aarC or gcpE.