The barrier function of the intestinal wall plays a key role in body homeostasis and defense against noxious agents. Conventional Ussing chamber techniques determine the overall transmural resistance but do not differentiate epithelial and subepithelial tissues. The barrier function, however, resides in the epithelial cell layer only. Transmural impedance analysis can solve this problem, if adequate models are applied. We show that: (i) epithelial and subepithelial impedances are additive, (ii) the epithelium proper can be represented by a very general electrical model, which demonstrates short-circuiting at high frequencies (due to cell membrane capacitances), and (iii) the reactance of subepithelial tissue can be described phenomenologically. Using an empirical expression for description of the subepithelial impedance, the present method allows the determination of the epithelial and the subepithelial resistance. This was exemplified in rat ileum, which defied adequate impedance analysis so far. Of the transmural DC resistance of 61 +/- 5 omega.cm2 (n = 8) the subepithelial contribution was 28 +/- 2 omega.cm2 and the epithelial resistance was 33 +/- 4 omega.cm2.