Objective: Epithelial cell layers transport solutes via transcellular and paracellular pathways. Conventional resistance (TER) measurements are insufficient to separate and quantify these two pathways. It was therefore the objective of the present study to develop a technique that allowed separate determination of para- and transcellular resistances (R^para, R^trans). Methods: Two-path impedance spectroscopy is based on transepithelial impedance measurement in specialized Ussing chambers, combined with a Ca²⁺-dependent modulation of R^para through EGTA and parallel flux measurements of a paracellular marker substance. Results: In HT-29/B6 colon cells and MDCK C7 and C11 kidney tubule cells, fluorescein was found to meet the criteria of a paracellular marker which varies in parallel to 1/R^para. In these cell lines, a reduction in the extracellular Ca²⁺ concentration altered R^para without affecting R^trans. Furthermore, exact knowledge of the resistance of subepithelial components was found to be essential. In MDCK C7 and C11 two-path impedance spectroscopy was successfully applied to determine changes in paracellular resistance after stable transfection with various tight junction proteins. In contrast, in Caco-2 colon cells fluorescein was transported also transcellularly so that its flux did not vary in parallel to 1/R^para. This indicates that the pre-conditions of the method have to be carefully evaluated for each new cell line and experimental condition. Conclusion: Two-path impedance spectroscopy is a new and valid technique to independently measure paracellular and transcellular resistance in epithelial cell lines.