Stimulations of cell growth and macromolecular synthesis of HeLa cells by insulin and low density lipoprotein (LDL) were studied in relation to the effect of intracellular K+. After replacement of the culture medium by a chemically defined medium (K-CDM), addition of insulin plus LDL stimulated their growth. Protein synthesis was fast for the first 18 hrs. and then slowed to a constant rate with or without these agents. DNA synthesis began to increase from 15 hrs., attaining a maximum at 18 hrs. After change from K-CDM to CDM containing RbCl (Rb-CDM), Rb+ replaced about 80% of the intracellular K+ in 2 hrs. Cell growth in Rb-CDM was very slow but was markedly enhanced by insulin plus LDL. No initially rapid protein synthesis was observed. DNA synthesis decreased with time, but addition of insulin plus LDL resulted in transient increase. Thus, the initial rapid protein synthesis in K-CDM may be a prerequisite for inducing DNA synthesis that stimulates subsequent cell growth. In Rb-CDM, insulin plus LDL stimulated cell growth by increasing DNA synthesis without changing the synthesis of bulk protein, implying that they induced synthesis of growth-related proteins.