The conjugative ability of the F plasmid of Escherichia coli is highly growth phase dependent, with plasmid transfer efficiency dropping rapidly as donor cells progress through the growth cycle towards stationary phase. Transfer is dependent on the expression of the plasmid transfer (tra) genes, which are controlled by three plasmid-encoded regulatory proteins: TraJ, TraY and TraM. Here, we show that the nucleoid-associated host protein, H-NS, acts to repress the expression of traM and traJ as cells enter stationary phase, thereby decreasing mating ability to barely detectable levels. Sequence analysis identified regions of predicted intrinsic curvature, to which H-NS preferentially binds, at the promoters of both traM and traJ. H-NS binding at these regions was then confirmed by electrophoretic mobility shift and DNase I protection footprinting assays. Immunoblot assays displayed a significant increase in TraJ and TraM levels in an hns mutant strain. These findings were further supported by Northern and primer extension analyses which showed that whereas both genes were only expressed in early exponential phase in wild-type cells, hns mutant cells exhibited drastic derepression throughout the growth cycle. Transcriptional fusion studies of the individual promoters demonstrated that H-NS-mediated repression was observed when the promoters of both traM and traJ were present in cis to each other. This suggests that H-NS may bind to an extended region of the F plasmid, acting as a regional silencer of promoters for traJ and traM.