β1 integrins are anchored on the basal membrane of enterocytes, but little is known about their localization in M cells, which are the main entry route into the intestinal mucosa for many bacterial pathogens. In particular, it has been suggested that adhesion of enteropathogenic Yersinia to M cells is mediated by interaction of the bacterial protein invasin and apical β1 integrins. Using a novel in vitro model of M cells, we demonstrate an augmented apical and basolateral targeting of β1 integrins in M cells associated with increased total α chain synthesis. The α3 and α6 subunits were targeted to the basal pole, but α2 subunit was targeted at both poles. No other α subunit was found associated with apical β1 integrins on M cells. Interestingly, Y. enterocolitica still adhered to the apical surface of M cells, despite the fact that α2β1 is not a receptor for invasin. We therefore studied the adhesive properties of invasin‐mutant Y. enterocolitica and invasin‐expressing Escherichia coli on the apical surface of M cells. We show that it is not invasin, but the product of an as yet unidentified bacterial chromosomal gene, that is involved in the adhesion of Y. enterocolitica to the apical membrane of M cells.