Novikoff rat hepatoma cells (subline N1S1-67) grew when 30 mM L-lactate or pyruvate was substituted for D-glucose in Swim's medium 67 supplemented with dialyzed calf bovine serum. A 2.6-fold increase in cell number (1.34 generations) was obtained. RNA, DNA, protein and dry weight increased in proportion to the cell number. In control medium lacking L-lactate, pyruvate or D-glucose, cell growth of 0.42 generation was obtained. Growth with L-lactate was dependent on the L-lactate concentration up to 30 mM at which the greatest increase in cell number occurred. Significant growth did not occur when D-lactate, glycerol, acetate, alpha-ketoglutarate, succinate or malate, each at 30 mM, was substituted for D-glucose. Growth in the medium containing L-lactate was not due to the utilization of D-glucose or some other substrate carried into the culture with the inoculum. Medium contamination by D-glucose was insufficient to explain the growth obtained in the medium containing L-lactate, but could have accounted for growth in the control medium. Throughout growth, the concentration of L-lactate in the medium remained unchanged. The increase in cell number cannot be explained by L-lactate triggering the utilization of glycogen, nor by oxidation and degradation of protein, amino acids, fatty acids, or carbohydrate moieties of glycoprotein in the medium. L-Lactate does not serve as a significant carbon or energy source in the growth of these cells.