Rat hepatocytes were freshly prepared from adult animals using the collagenase-perfusion technique. The hepatic transport of thiamine was studied in isolated liver cells. The process was found to be saturable with an apparent Kt of 0.31 mM and a V max of 0.7 mumoles/ml intracellular fluid/5 minutes. However, at higher substrate concentrations, the process proceeded in a linear fashion. Both pyrithiamine and oxythiamine were inhibitory on the hepatic uptake of thiamine, the latter showed much weaker activity than the former. The system required the presence of sodium ions and was sensitive to ouabain. Anaerobic condition and metabolic inhibitors, e.g., 2,4-dinitrophenol, cyanide, and iodoacetate suppressed the uptake rate of thiamine. Addition of ethanol in the incubation medium also caused significant reduction of thiamine uptake. Efflux studies indicated that a portion of intracellular thiamine is readily available for exodus. Chromatographic analyses showed that thiamine was only slightly metabolically altered during the transport process. It is suggested that thiamine is transported into isolated hepatic cells by an active, sodium-dependent process.