Butyrobetaine transport into the liver was studied using isolated rat hepatocyte plasma membrane vesicles. In the presence of a sodium chloride gradient, an overshoot could be observed, indicating active sodium-dependent transport. A similar overshoot was recorded in the presence of lithium, but not of potassium, cesium, or choline chloride. Investigation of several sodium salts revealed that an overshoot could only be observed in the presence of chloride, but not of nitrate, thiocyanate, sulfate, or gluconate. An osmolarity plot in the presence of sodium chloride revealed a slope different from zero and a positive intercept, indicating active transport and nonspecific binding, respectively. In agreement with the osmolarity plot, the kinetic characterization of butyrobetaine transport revealed a binding and a saturable component. The saturable component could be described by Michaelis-Menten kinetics, with a Kmof 4.88 ± 0.70 mmol/L and a Vmaxof 4.16 ± 0.73 picomoles per milligram of protein per second. Butyrobetaine transport could be inhibited significantly (30%) by 250 μmol/L propionylcarnitine, but not by d - or l -carnitine, other acylcarnitines (acetylcarnitine, isovalerylcarnitine, palmitoylcarnitine), trimethyllysine, or quinine. Butyrobetaine transport activity was also expressed in Xenopus laevis oocytes by injecting mRNA isolated from rat liver or kidney. After 5 days of cultivation, the endogenous butyrobetaine transport activity was increased by 82% in oocytes injected with liver mRNA and by 99% in oocytes injected with kidney mRNA. The studies show that butyrobetaine is transported actively across the basolateral plasma membrane of hepatocytes and that this transport is driven by sodium and chloride gradients. This transport is quite specific for butyrobetaine and is not rate-limiting for carnitine biosynthesis.