Plasma membrane fractions were prepared from homogenates of rat liver by density gradient centrifugation and then used for the formation of right-side-out vesicles. Uptake of Pi into the vesicles is rapid when an inwardly directed sodium gradient is present and an overshoot of uptake occurs indicative of accumulation against a Pi concentration gradient. Initial Pi uptake rate in the presence of a K+ gradient is approx half that seen with Na+, but uptake in the presence of a choline chloride gradient is very slow. An overshoot does not occur with either K+ or choline gradients. The Km(Pi) for the Na-dependent component of Pi uptake is approx. 1 mM and Vmax at 20 degrees C is 0.8 nmol/min per mg protein. The relationship between initial uptake rate and Na+ concentration is sigmoid, with a Hill coefficient of 2.6. It is concluded that the cotransporter resembles that of kidney and intestine in possessing at least two interacting sites for Na+ and that in intact cells the Na+ gradient maintained by the sodium pump ATPase provides the energy for accumulation of Pi against the unfavourable membrane potential.