Aims: To examine the involvement of N-acyl homoserine lactone (AHL) quorum sensing in alkaline metalloprotease (aprX) promoter regulation in a Pseudomonas fluorescens milk isolate. Methods and Results: N-acyl homoserine lactone signals from P. fluorescens strain 395 were separated and detected by thin layer chromatography (TLC). Further analysis of the AHL signals using liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) indicated the presence of C4-HSL and 3OC8-HSL. The expression of aprX in P. fluorescens 395 was investigated, using a transcriptional fusion between the aprX promoter and a mutated gfp variant gene (gfp[mut3]). The results demonstrated that the activity of the aprX promoter increased dramatically in the late exponential phase of growth, indicating growth phase-dependent regulation. The activity was repressed in an AHL-deficient environment, in which the signal molecules were hydrolysed by the enzyme AHL lactonase. Conclusions: N-acyl homoserine lactones produced by P. fluorescens 395 were identified to be C4-HSL and 3OC8-HSL. The protease gene in P. fluorescens is regulated by the AHL-based quorum sensing system at a transcriptional level during the late exponential growth phase. Significance and Impact of the Study: This study contributes to our understanding of the genetic regulation and ecology of P. fluorescens in the economically important system of food spoilage.