When using cultured cell monolayers to determine the mechanism of transcellular diffusion of molecules, it may be important to identify the fraction that moves through the paracellular route or passively diffuses through tight junctions. We characterized the apparent diameter of the junctional pore in a variety of epithelial cell monolayers (Caco-2, MDCK, alveolar). Using hydrophilic extracellular permeants varying in molecular radii and charge (neutral, anionic, cationic, zwitterionic), rate-determining steps and factors of the paracellular route were quantitatively delineated by the model for molecular size-restricted diffusion within a negative electrostatic field of force. Protonated amines permeated the pores faster than their neutral images while organic anions were slower. With increasing molecular size the influence of charge diminished. This approach was used to quantify the relationship between permeant radius and transepithelial electrical resistance and to analyze changes in junctional pore size as a function of pharmacological perturbation, such as in the use of absorption promoters or adjuvants.