We applied the principles of molecular-size-restricted diffusion within a negative electrostatic field of force to follow the changes in the aqueous pore radius of tight junctions (TJs) induced by perturbants and the accompanying influence on the permeation of neutral (urea and mannitol), cationic (methylamine and atenolol), and anionic (formate and lactate) compounds that vary in size. The perturbants included palmitoyl-DL-carnitine (PC), which opens TJs by an unknown Ca++-independent mechanism, and ethyleneglycol-bis-(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), a Ca++ chelator. Mass transfer resistances of the collagen-coated filter support and the aqueous boundary layers were factored out to yield paracellular permeability coefficients (P[P]). As viewed from the P(P) values of urea and mannitol, EGTA exhibited insignificant effects on pore size at low concentrations compared with control, and then caused a dramatic opening of the TJs over a narrow concentration range (1.35-1.4 mM). The P(P) values for urea and mannitol remained constant at >1.4 mM EGTA. However, PC produced dose-dependent responses from O to 0.15 mM that plateaued at >0.15 mM. In general, cations permeated the cellular TJs faster and anions slower than their neutral images. The effects of changes in pore size (4.6 to 14.6 A in effective radius) on the ability of these solutes to permeate the TJs were analyzed by the Renkin molecular sieving function. These studies established an experimental, theoretical, and quantitative template to assess perturbants of the TJ and define the limits, short of detrimental effects, at which the TJs may be sufficiently perturbed for maximal enhancement of permeation of solutes varying in size and charge.