Phosphofructokinase activity increased dramatically in cultured mouse spleen lymphocytes 8 hours after concanavalin A stimulation and preceded the onset of DNA synthesis by 8 hours. The increase in enzyme activity and [3H]-thymidine incorporation were mitogen-concentration dependent. Enzyme activity increased 12-fold over control level at 48 hours when DNA synthesis peaked. The protein synthesis inhibitor, cycloheximide, blocked the rise in phosphofructokinase only when given prior to the increase in enzyme activity. Once the increase began, later addition of cycloheximide became progressively less inhibitory. These observations suggest that the period of increase in phosphofructokinase activity involves the activation of preexisting enzyme molecules.