Photorhabdus luminescens is an insect-pathogenic bacterium that forms a symbiosis with specific entomopathogenic nematodes. In this bacterium, a symbiosis-'deficient' phenotypic variant (known as the secondary variant or form II) arises at a low frequency during prolonged incubation. A knock-out mutant was generated of the regulator of a newly identified two-component regulatory system, designated AstR-AstS. Interestingly, this mutation altered the timing of phenotypic switching. Variant cells arose in the mutant strain several days before they did in the wild-type population, suggesting that AstRS is directly or indirectly involved in the genetic mechanism underlying variant cell formation. This mutation also affected motility and antibiotic synthesis. To identify AstRS-regulated genes, a comparative analysis using two-dimensional gel electrophoresis was performed. Seventeen proteins with modified synthesis in stationary phase were identified by mass spectrometry and shown to be involved in electron-transport systems, energy metabolism, iron acquisition and stress responses. The results imply that AstRS is involved in the adaptation of cells to the stationary phase, whilst negatively affecting the competitive advantage of form I cells. The link between AstRS-dependent stationary-phase adaptation and phenotypic variation is discussed.