Addition of serum to quiescent cultures of 3T3 cells rapidly increases lactic acid formation and subsequently stimulates cell division. The stimulation of lactic acid production is seen at high, saturating concentrations of extra-cellular glucose. It is dependent on the time of exposure and on the dose of serum and is not blocked by the addition of cycloheximide, puromycin, or actinomycin D. In contrast, serum only marginally affects glycolysis by rapidly growing 3T6 or SV40-3T3 cells. In addition to serum, epidermal growth factor (0.1 to 10 ng/ml) and insulin (10 to 500 ng/ml) cause a striking stimulation of glycolysis in quiescent 3T3 cells. Neither exogenous cyclic nucleotides nor ouabain effect the glycolytic response, but the presence of Ca2+ markedly influences the activation of glycolysis by epidermal growth factor and by insulin. A novel finding in this study is that homogenates prepared from quiescent cells treated with serum, epidermal growth factor, or insulin show increased glycolysis as compared with homogenates from nonstimulated cultures. This finding will allow further experimental analysis of the cause of increased glycolysis in rapidly proliferating cells.