The mechanism by which cells take up retinol from retinol-binding protein (RBP) and the role of the RBP-transthyretin (TTR) complex remain unclear. Here we report on retinol uptake through the RBP-TTR complex by primary cultured rat hepatocytes (parenchymal cells, PC) and nonparenchymal cells (NPC) following incubation with [3H]retinol-RBP or the [3H]retinol-RBP-TTR complex under several conditions. The cellular accumulation of retinol was time and temperature dependent in both PC and NPC. Analysis by HPLC showed that the incorporated [3H]retinol in NPC was mainly converted to retinyl ester, although in PC it remained mainly as unesterified retinol. However, the amount of retinol taken up from the RBP-TTR complex was nearly twofold greater than that from RBP alone. The uptake of [3H]retinol from protein-bound retinol was inhibited by an excess of either retinol-RBP or retinol-RBP-TTR complex. Moreover, retinol uptake through the RBP-TTR complex was inhibited by an excess of free TTR. From these results we postulate that TTR may take part as a positive regulator in the delivery of RBP-bound retinol from plasma, possibly by a membrane receptor, and that retinol uptake takes place preferentially from the RBP-TTR complex into both PC and NPC. The uptake of [3H]retinol (2 microM) by PC was saturated, whereas uptake by NPC was not. These results indicate that the physiological importance of TTR in retinol delivery may be especially important to vitamin A-storing stellate (Ito) cells in the NPC fraction.