This study has evaluated an in vitro model of the follicle-associated epithelia that overlie Peyer's patches of the small intestine. The model shares many phenotypic characteristics of M- cells in vivo. Co-cultures of the human adenocarcinoma cell line Caco-2 and freshly isolated Peyer's patch cells were established. Fluorescence microscopy and quantitative image analysis were used to validate the model against known markers of M-cell phenotype. Apical expression of alkaline phosphatase was down-regulated in co-cultures and villin was re- distributed from the apical membrane to the cytoplasm. a5ß1 integrin was found on the apical surfaces of the monolayers and B and T lymphocytes integrated into the monolayers. Particle transport was temperature-dependent in co-cultures, indicating that a transcytotic route was responsible. This model provides opportunities to study factors that influence M-cell development, assess putative Peyer's patch targeting in oral vaccine technologies, and study intestinal uptake in vitro.