Since excessive epidermal hyaluronan is associated with hyperproliferative states and disturbed terminal differentiation of the keratinocytes, we hypothesized that 4-methylumbelliferone (4-MU), an inhibitor of hyaluronan synthesis, could counteract these phenotypic features. Cultured epidermal keratinocytes showed a concentration dependent, maximum 83% reduction of hyaluronan in the presence of 0.2–1.0 mM 4-MU, whereas less decline was seen in the synthesis of chondroitin and heparan sulfate. The reduced hyaluronan was associated with no apparent change in its molecular mass. The 4-MU-treated keratinocytes showed an accentuated epithelial morphology with a flat, round cell shape, increased stress fibers and large vinculin-positive adhesion plaques, cytoskeletal changes consistent with the markedly reduced migration rate observed in scratched monolayer cultures. High concentrations of 4-MU also caused a block in keratinocyte proliferation, reversible upon 4-MU withdrawal. In the epidermis of organotypic cultures, 4-MU prevented the hyaluronan accumulation and epidermal hypertrophy induced by epidermal growth factor. The present results concur with earlier data indicating that enhanced cell locomotion and proliferation are associated with hyaluronan synthesis in activated keratinocytes. Cell proliferation, however, was blocked more strongly than expected on the basis of the incomplete hyaluronan synthesis inhibition, and may represent a novel target of 4-MU. At any rate, 4-MU and equivalent hyaluronan synthesis inhibitors might be considered for situations where suppression of epidermal activation and hyperproliferation is warranted.