Decreases in plasma folate levels leading to folate deficiency can result from increased urinary loss of folate, due to changes in either the renal reabsorption or secretion of folate. Hence, human proximal tubule (HPT) cells were cultured on microporous membranes to separate apical (AP) and basolateral (BL) domains and to assess the transport of 5-methyltetrahydrofolate from AP-to-BL (i.e., reabsorptive) and BL-to-AP (secretory) directions. Cellular uptake of alpha-methylglucoside occurred specifically from the AP direction, and transport of p-aminohippurate occurred more readily from the BL direction, demonstrating cell polarity similar to that in vivo. Under tight monolayer conditions, binding of folate to the AP membrane occurred more readily from the AP direction, although AP binding also occurred from the BL chamber. Intracellular transport occurred equally from both AP and BL directions. When loaded from either direction, folate was effluxed from HPT cells into both AP and BL chambers. About 20-30% of the internalized substrate was converted to nonfolate catabolites. Thus HPT cells readily take up folate via both the AP and BL membranes, metabolize it intracellularly and secrete the products across both membranes. These studies suggest that renal folate homeostasis is regulated bidirectionally.