The aim of this study was to characterize the in vitro hepatic uptake kinetics of sodium fluorescein (NaFluo) and identify the transporters involved. NaFluo exhibited saturable uptake kinetics in suspended rat and human hepatocytes as reflected by K(m) values of 22.5 and 14.1 µM, and V(max) values of 98.3 and 5.8 pmol/(million cells • min), respectively. Coincubation with known inhibitors (e.g. rifampicin) of organic anion transporting polypeptide (OATP/Oatp; SLCO gene family) significantly decreased NaFluo uptake in hepatocytes. In contrast, neither inhibitors/substrates of the organic cation transporter or organic anion transporter family nor depletion of extracellular sodium resulted in significant inhibition of NaFluo uptake. To explore the contribution of individual uptake transporters, NaFluo uptake was determined in Chinese hamster ovary cells transfected with OATP1B1, OATP1B3, and OATP2B1. Transporter-mediated uptake of NaFluo was observed in OATP1B1- and OATP1B3-transfected cells (K(m) = 4.2 and 10.9 µM; V(max) = 30.9 and 135 [pmol/(mg protein • min)], respectively). NaFluo can be used as a probe substrate to study Oatp/OATP1B-mediated drug interactions in fluorescence-based in vitro transport assays of rat and human liver. Labeling of drugs or bile salts with a fluorescein moiety can be expected to result in fluorescent conjugates with substantially altered hepatic uptake characteristics as compared with the unconjugated compounds.