Type III secretion-mediated cytotoxicity is one of the key virulence mechanisms of the opportunistic pathogen Pseudomonas aeruginosa. Prior data from several laboratories have established that metabolism is a key factor in the regulation of type III secretion gene expression in P. aeruginosa. Here we use a fluorescence-activated cell sorter (FACS)-based approach to investigate expression of type III secretion genes at a single-cell level. The data demonstrate that the metabolic state regulates the percentage of cells that are able to induce type III secretion gene expression under inducing conditions. We also present evidence that this regulation is the result of an effect of the growth conditions on the ability of P. aeruginosa to assemble a functional type III secretion apparatus. Preliminary data suggest that the metabolite that controls type III secretion gene expression is derived from acetyl-CoA and that this regulation may, in part, be mediated by changes in the intracellular concentration of cyclic-AMP.