Pseudomonas fluorescens Pf-5, a rhizosphere bacterium, produces a suite of secondary metabolites that are toxic to seed- and root-rotting plant pathogens. Among these are the polyketide compounds pyoluteorin and 2,4-diacetylphloroglucinol. We provide evidence that pyoluteorin production is influenced by positive autoregulation. Addition of pyoluteorin to liquid cultures of Pf-5 enhanced pyoluteorin production. In addition, pyoluteorin and 2,4-diacetylphloroglucinol mutually inhibit one another's production in Pf-5. For pyoluteorin, both positive autoregulation and negative influences on production by 2,4-diacetylphloroglucinol were demonstrated at the transcriptional level by measuring activity from transcriptional fusions of an ice nucleation reporter gene (inaZ) to three separate pyoluteorin biosynthetic genes. The occurrence of pyoluteorin autoregulation in the rhizosphere was assessed on cucumber seedlings in pasteurized soil with cross-feeding experiments. In the rhizosphere, expression of a pyoluteorin biosynthesis gene by a pyoluteorin-deficient mutant of Pf-5 was enhanced by pyoluteorin produced by coinoculated cells of Pf-5. These data establish that the polyketide pyoluteorin is an autoregulatory compound and functions as a signal molecule influencing the spectrum of secondary metabolites produced by the bacterial cell.