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Virulence factors generally enhance a pathogen's fitness and thereby foster transmission. However most studies of pathogen fitness have been performed by averaging the phenotypes over large populations. Here we have analyzed the fitness costs of virulence factor expression by Salmonella enterica subspecies I serovar Typhimurium in simple culture experiments. The type III secretion system ttss 1 a cardinal virulence factor for eliciting Salmonella diarrhea is expressed by just a fraction of the S. Typhimurium population yielding a mixture of cells that either express ttss 1 (TTSS 1(+) phenotype) or not (TTSS 1( ) phenotype). Here we studied in vitro the TTSS 1(+) phenotype at the single cell level using fluorescent protein reporters. The regulator hilA controlled the fraction of TTSS 1+ individuals and their ttss 1 expression level. Strikingly cells of the TTSS 1(+) phenotype grew slower than cells of the TTSS 1( ) phenotype. The growth retardation was at least partially attributable to the expression of TTSS 1 effector and/or translocon proteins. In spite of this growth penalty the TTSS 1(+) subpopulation increased from