We examined whether [131I]6-β-iodomethyl-19-norcholesterol (NP-59), a cholesterol analog, can be used to measure function of hepatic drug transporters. Hepatic uptake of NP-59 with and without rifampicin was evaluated using HEK293 cells expressing solute carrier transporters. The stability of NP-59 was evaluated using mouse blood, bile, and liver, and human liver S9. Adenosine triphosphate-binding cassette (ABC) transporters for bile excretion were examined using hepatic ABC transporter vesicles expressing multidrug resistance protein 1, multidrug resistance-associated protein (MRP)1-4, breast cancer resistance protein (BCRP), or bile salt export pump with and without MK-571 and Ko143. Single photon emission computed tomography (SPECT) was performed in normal mice injected with NP-59 in the presence or absence of Ko143. Uptake of NP-59 into HEK293 cells expressing organic anion transporting polypeptide (OATP)1B1 and OATP1B3 was significantly higher than that into mock cells and was inhibited by rifampicin. NP-59 was minimally metabolized in mouse blood, bile, and liver, and human liver S9 after 120 min of incubation. In vesicles, NP-59 was transported by MRP1 and BCRP. Excretion of NP-59 into bile via BCRP was observed in normal mice with and without Ko143 in the biological distribution and SPECT imaging. NP-59 can be used to visualize and measure the hepatic function of OATP1B1, OATP1B3, and BCRP.