The prevalence of disinfectant-resistant food-related microorganisms is of concern to the food industry. The Staphylococcus saprophyticus strain ST2H6 isolated from a poultry processing plant contained a 2.4-kb plasmid (p2H6) harbouring qacH, which encodes resistance to disinfectants based on quaternary ammonium compounds. The complete p2H6 nucleotide sequence revealed an open reading frame encoding a putative protein of 107 amino acid residues with strong similarity to members of the small multidrug resistance protein family. QacH also conferred high-level ethidium bromide resistance and low-level proflavine resistance and thus differed phenotypically from the similar proteins Smr and QacG. Fluorimetry indicated that the high-level ethidium bromide resistance was due to improved efflux energised by the proton motive force. Site-directed mutagenesis substituting the Asp-24 residue with Glu-24 had no effect on resistance characteristics. An additional open reading frame on p2H6 encoded a putative protein with similarity to rolling circle replication proteins.