Triggering the ceramide pathway by exogenous treatment with neutral sphingomyelinase (Smase) inhibited human keratinocyte growth rate, while having no influence on cell apoptosis. Increasing the ceramide content of keratinocytes with Smase (100 U/ml) or C6-ceramide (1 microM) enhanced matrix metalloproteinase (MMP)-9 production. On the contrary, levels of MMP-2 secretion were unchanged. The inhibition of keratinocyte growth rate induced by ceramide could be annihilated by a peptide hydroxamate MMP inhibitor or an MMP-9 blocking antibody. In addition, inhibiting MMP-9 activity in control keratinocyte culture was found to stimulate keratinocyte proliferation. These data suggest a pivotal function of MMP-9 in the control of keratinocyte growth.