A431 cells were treated with epidermal growth factor (EGF) to study the mechanism by which this factor accelerates the glycolytic flux. After EGF treatment, fructose-2,6-bisphosphate (Fru-2,6-P2) levels rose up to 2-fold. This change correlated with an increase in phosphofructokinase-2 activity, which was not due to a change in the transcription or translation of the enzyme, neither in the amount of enzyme. PK-C does not appear to be involved in the signalling mechanism since EGF was equally potent in PK-C depleted cells than in control cells. The increase in Fru-2,6-P2 levels was lower and more transient in cells treated with EGF in a calcium-free medium than in the presence of the cation, and it was restored by the addition of calcium to the medium. These results suggest a possible role for calcium-mediated pathways in the control of Fru-2,6-P2 levels in A431 cells. © 1992 Academic Press, Inc.