A role for the internal elastic lamina (IEL), which separates the intima and media of an artery wall, as a restrictive barrier to macromolecular movement has been suggested in atherosclerotic lesion development or restenosis during angioplasty. The permeability coefficient of the IEL, however, has never been quantified in unperturbed vessels in vivo. Using a newly developed technique, we measured the concentration distributions in both intima and media of cationic (pI ≈ 8.5) and anionic (pI ≈ 6.3) isozymes of the 44-kD macromolecule horseradish peroxidase (HRP). Two mathematical models of arterial wall transport differing in their resolution of the intima were required to simulate the concentration distribution data and to estimate the parameters of interest. Optimal estimates of the permeability coefficients of the endothelium (PE) and IEL (PIEL) to HRP were determined by the best least-squares fit of the two models to experimental data. These estimates (anionic: PE =0.050±0.021 μm/min, PIEL = 0.146±0.082 μm/min, n = 8; cationic: PE = 0.034 ± 0.018 μm/min, PIEL = 0.110±0.047 μm/min, n = 8) indicate that the IEL is responsible for ≈25% (anionic, 26±9%; cationic, 25±13%) of the resistance to HRP transport from the blood into the arterial media. Although both parameters were less for the cationic preparation, the differences were not significant, and the relative role of the IEL was similar for both molecules. These data demonstrate the importance of the IEL in controlling the intimal accumulations of plasma-borne macromolecules, and they imply a role for the IEL in influencing paracrine communication between cells of the intima and media.