Primary cultures of rat hepatocytes take up phosphate by a saturable Na(+)-dependent process. Thus the plasma membrane possesses an N(+)-Pi cotransporter of the type described for many cell types, e.g., kidney proximal tubular cells and enterocytes. Coupling to Na+ overcomes the barrier to anion entry represented by the membrane potential. At 0.12 mM Pi, the effect of Na+ is cooperative with a Hill coefficient of 1.7 suggesting two sodium sites per molecule of carrier. At 37 degrees C, the Km (for Pi) and Vmax for the sodium-dependent fraction of Pi uptake are approx. 1 mM and 0.35 nmol Pi/min per mg cell protein, respectively. Insulin stimulates Vmax four-fold with no significant effect on Km. Pi uptake in the absence of sodium is not affected by insulin. The stimulation by insulin could be of metabolic significance. Glucose phosphorylation at the expense of ATP is raised in liver following insulin stimulation, and thus, initially there may be an increased demand for Pi for oxidative phosphorylation until new steady-state conditions of hexose phosphate concentrations and of ATP turnover become established.