The transendothelial transfer of macromolecules has been difficult to study because of the complexities of the in vivo models. We have developed a model of an endothelium cultured on a permeable support and used it to characterize the transendothelial transfer of albumin. Porcine pulmonary artery endothelial cells form a single layer of cells lining the gelatin-impregnated polycarbonate micropore filters, and the cells develop junctional structures similar to endothelial tight junctions observed in vivo. The monolayer resists the flow of electrical current, and the resistance is sensitive to extracellular calcium concentrations. Albumin transfer across the cultured monolayers was found to be asymmetric, and the rate of transfer from interstitium to lumen was greater than that from lumen to interstitium. The asymmetric transfer occurred against a concentration gradient and was abolished by treating the monolayer with NaCN. Increasing albumin concentrations increased the rate of interstitial to luminal transfer, and the process demonstrated saturation at an interstitial albumin concentration of 725 microM. These data point out the usefulness of the in vitro preparation to identify potentially important aspects of transendothelial transport that would be difficult to detect in vivo.