Permeable membranes are indispensable for in vitro epithelial barrier models. However, currently available polymer-based membranes are low in porosity and relatively thick, resulting in a limited permeability and unrealistic culture conditions. In this study, we developed an ultrathin, nanoporous alumina membrane as novel cell culture interface for vertebrate cells, with focus on the rainbow trout (Onchorynchus mykiss) intestinal cell line RTgutGC. The new type of membrane is framed in a silicon chip for physical support and has a thickness of only 1 mu m, with a porosity of 15% and homogeneous nanopores (empty set = 73 +/- 21 nm). Permeability rates for small molecules, namely lucifer yellow, dextran 40, and bovine serum albumin, exceeded those of standard polyethylene terephthalate (PET) membranes by up to 27 fold. With the final goal to establish a representative model of the fish intestine for environmental toxicology, we engineered a simple culture setup, capable of testing the cellular response toward chemical exposure. Herein, cells were cultured in a monolayer on the alumina membranes and formed a polarized epithelium with apical expression of the tight junction protein ZO-1 within 14 days. Impedance spectroscopy, a noninvasive and real time electrical measurement, was used to determine cellular resistance during epithelial layer formation and chemical exposure to evaluate barrier functionality. Resistance values during epithelial development revealed different stages of epithelial maturity and were comparable with the in vivo situation. During chemical exposure, cellular resistance changed immediately when barrier tightness or cell viability was affected. Thus, our study demonstrates nanoporous alumina membranes as promising novel interface for alternative in vitro approaches, capable of allowing cell culture in a physiologically realistic manner and enabling high quality microscopy and sensitive measurement of cellular resistance.