The hepatic extraction of a naturally occurring secosteroid, vitamin D3 (D3), in relation to its hepatic arterial or portal venous route of delivery has been studied in isolated rat liver preparations perfused at an arterial/venous flow ratio of 1:4. No significant difference in the fractional hepatic extraction of D3 was observed when the vitamin was administered via the portal venous route compared to when it was administered via the hepatic arterial route. Estimation of the uptake and clearance of D3 in relation to its route of delivery revealed, however, that due to the higher perfusion flow through the portal venous than through the arterial route, both the hepatic uptake and clearance of D3 were significantly higher after portal vein than after hepatic artery delivery. Moreover, calculation of the uptake of D3 after delivery through the portal venous route also revealed that it was not significantly different from that of the total hepatic uptake (uptake following portal vein + hepatic artery delivery). The data obtained during the present studies indicate, then, that the fractional hepatic extraction of D3 is not dependent on its route of entry into the liver; it also points out that, in experimental models such as in isolated-perfused liver preparations, the portal vein administration of D3 should represent adequately the total hepatic handling of the secosteroid by the normal rat liver.