Uptake of lucifer yellow (LY), a fluorescent disulfonic acid anionic dye, was studied in isolated skate (Raja erinacea) perfused livers and primary hepatocytes to evaluate its utility as a fluid-phase marker in these cells. However, our findings demonstrated that LY is transported across the plasma membrane of skate hepatocytes largely via carrier-mediated mechanisms. Isolated perfused skate livers cleared 50% of the LY from the recirculating perfusate within 1 h of addition of either 22 or 220 µM LY, with only 4.5 and 9% of the LY remaining in the perfusate after 7 h, respectively. Most of the LY was excreted into bile, resulting in high biliary LY concentrations (1 and 10 mM at the two doses, respectively), indicating concentrative transport into bile canalicular lumen. LY uptake by freshly isolated skate hepatocytes was temperature sensitive, exhibited saturation kinetics, and was inhibited by other organic anions. Uptake was mediated by both sodium-dependent [Michaelis-Menten constant (K_m), 125 ± 57 µM; maximal velocity (V_max), 1.5 ± 0.2 pmol · min¹ · mg cells¹] and sodium-independent (K_m, 207 ± 55 µM; V_max, 1.7 ± 0.2 pmol · min¹ · mg cells¹) mechanisms. Both of these uptake mechanisms were inhibited by various organic anions and transport inhibitors, including furosemide, bumetanide, sulfobromophthalein, rose bengal, probenecid, N-ethylmaleimide, taurocholate, and p-aminohippuric acid. Fluorescent imaging techniques showed intracellular vesicular compartmentation of LY in skate hepatocyte clusters. Studies in perfused rat livers also indicated that LY is taken up against a concentration gradient and concentrated in bile. LY uptake in isolated rat hepatocytes was saturable, but only at high concentrations, and demonstrated a K_m of 3.7 ± 1.0 mM and a V_max of 1.75 ± 0.16 nmol · min¹ · mg wet wt¹. These results indicate that LY is transported into skate and rat hepatocytes and bile largely by carrier-mediated mechanisms, rather than by fluid-phase endocytosis.