Transport of an organic anion, p-aminohippurate (PAH), was investigated in an established cell line derived from the American opossum kidney (OK). Transepithelial transport and accumulation of PAH were measured in OK cells cultured on permeable supports. Specifically mediated transcellular transport of PAH across OK cell monolayers occurred only from basal to apical side, and the accumulation of PAH in the cells was much larger from basal side than from apical side. Both the transcellular transport of PAH from basal to apical side and the accumulation from basal side were temperature dependent, saturable, and inhibited by organic anions such as probenecid but not by organic cations. In contrast, such a specific transport of PAH was not observed in LLC-PK1, another epithelial cell line derived from pig kidney. These findings suggest that OK cells can transport PAH by the organic anion transport system unidirectionally from basal to apical side across the cell monolayers and that OK cells might be a useful model for the study of organic anion secretion in the renal proximal tubules.