One of the major mechanisms that plant growth-promoting bacteria use to facilitate plant growth is through the lowering of plant ethylene levels by the bacterial enzyme 1-aminocyclopropane-1-carboxylate (ACC) deaminase. Many of the bacterial ACC deaminase genes (acdS) that have been examined to date are under the transcriptional control of a leucine-responsive regulatory protein, Lrp, encoded by acdR and referred to here as AcdR. The work presented here is focused on how AcdR and the newly discovered AcdB protein from Pseudomonas putida UW4 are involved in the regulation of acdS expression. First, the results of gel retardation experiments showed that AcdR binds to the acdS regulatory region, and this binding activity in vitro is not affected by the addition of 2 mmol·L–1ACC but can be eliminated by addition of 20 μg·mL–1leucine. Second, a potential regulatory protein, AcdB, involved in the regulation of acdS expression, was identified through both yeast 2-hybrid screen and coimmunoprecipitation based on its ability to bind to AcdR; subsequently, its binding to the acdS regulatory region in the presence of ACC was shown by gel retardation experiments. The data are interpreted in terms of a model in which AcdR and AcdB co-regulate the expression of the acdS gene.