Acinetobacter calcoaceticus PHEA-2 exhibited a delayed utilization of phenol in the presence of benzoate. Benzoate supplementation completely inhibited phenol degradation in a benzoate 1,2-dioxygenase knockout mutant. The mphR encoding the transcriptional activator and mphN encoding the largest subunit of multi-component phenol hydroxylase in the benA mutant were significantly downregulated (about 7- and 70-fold) on the basis of mRNA levels when benzoate was added to the medium. The co-transformant assay of E. coli JM109 with mphK::lacZ fusion and the plasmid pETR carrying mphR gene showed that MphR did not activate the mph promoter in the presence of benzoate. These results suggest that catabolite repression of phenol degradation by benzoate in A. calcoaceticus PHEA-2 is mediated by the inhibition of the activator protein MphR.