The mouse hepatoma cell (Hepa-1) in tissue culture has been shown to synthesize and secrete three electrophoretically distinct transferrins. Each of these forms of transferrin has a molecular weight of 77,000, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The concentration of each form is indicated by its staining intensity, which is highest in the form with the fastest mobility and lowest in the form with the slowest mobility. The relative rate of transferrin synthesis has been determined in log-phase and stationary-phase cells; the data indicate that the relative rate of synthesis increases twofold in stationary-phase cells. When the incorporation of [3H]leucine into transferrin reaches steady state, the rate of secretion is equal to the rate of synthesis; the rate of secretion also increases twofold in stationary-phase cells. Our studies also show that transferrin synthesis accounts for 0.98% of the total protein synthesis in log-phase cells and for 1.8% in stationary-phase cells. This is the level of synthesis that has been determined by in vivo studies. We conclude that after continuous culture for several years these hepatoma cells have maintained one of the characteristics of the differentiated liver cell, namely, the ability to synthesize and secrete transferrin.