Organotypic cultures have been used to study epithelial cell behavior for many years. Although current techniques produce multicellular epithelium that is able to differentiate, they lack properly organized basement membrane zone (BMZ). The aim of this study was to develop an organotypic culture method that better mimics the three dimensional morphology of interdigitating rete ridges and connective tissue (CT) papillae and conserves the BMZ. Bovine tongue mucosa was chosen for the raft donor and incubated with cold 1 M sodium chloride solutions for 4 days to separate the epithelial tissue from the BMZ and underlying CT matrix. Level of separation was studied by immunohistochemical staining of beta 1 and beta 4 integrins, laminin-1 and -5, type IV and VII collagen, tenascin, fibronectin, and heparan sulfate proteoglycan. After the separation, integrins were found exclusively at the basal cells of separated epithelium while other components of the BMZ were localized in the CT side suggesting that the separation occurs at the level of the lamina lucida. Rafts were rinsed several times with cell culture medium and used for growth organotypic cultures of human keratinocytes. Keratinocytes were cultured submerged in keratinocyte growth medium for 6 days after which the cultures were raised to air-liquid interface for up to 30 days. Cultures were terminated first after 5 days and weekly thereafter. Frozen sections were prepared and used for routine histology and immunohistochemistry (IHC). Stratification of keratinocytes was evident already after 5 days although the number of cell layers increased with time. Often more than 15 cell layers resembling normal epithelial histology were present. Keratinocytes in these raft cultures were found to express beta 4 integrins against the preexisting basement membrane components. In addition, human keratinocytes deposited their own basement membrane molecules, particularly laminin-1 and -5, on the preexisting bovine matrix. This culture model seems to mimic normal epithelium including the BMZ and is potentially useful for multiple applications for studies on epithelial cell behavior in vitro. Schlagwörter: cell-culture, organotypic, HaCat, basement membrane, differentiation