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The aim of this study was to use confocal scanning laser microscopy to examine the in situ localization of Escherichia coli O157:H7 on beef (knuckle or brisket) and carrots and in semisoft cheese made from pasteurized milk. Using a combination of specific immunolabeling and dual-excitation confocal scanning laser microscopy, it was possible to clearly demonstrate the localization of E. coli O157:H7 within various food types. In carrots, bacteria were found mainly at cell junctions and in intracellular spaces up to 50 microm deep. In beef, bacteria were located primarily between muscle fibers and within connective tissue (at a depth of 25 microm), whereas in cheese the bacteria occurred singly or in small clumps of up to 10 cells and were observed within the protein matrix of the cheese. These results revealed how E. coli O157:H7 can penetrate beef and carrot surfaces where it is protected from decontamination processes.