The aim of this work was to clarify the reason why a discrepancy exists between the effects of epidermal growth factor (EGF) on fibroblasts in culture repressing collagen biosynthesis and in vivo stimulating wound healing. The effect of EGF on the biosynthetic activity of fibroblasts was measured in various conditions of cultures: on plastic, on plastic coated with various macromolecules of the extracellular matrix, on top of a type I collagen gel, and within a three-dimensional collagen lattice. While the noncollagen protein (NCP) synthesis was not affected by the interactions of the cell with the various coated matrices, collagen synthesis was inhibited. At the surface of a collagen gel, protein synthesis was reduced, while collagen synthesis and degradation were slightly stimulated. When embedded in a lattice, the overall biosynthetic activity of fibroblasts was largely depressed. The addition of EGF to cultures on plastic and on the various coated macromolecules resulted in a further repression of collagen synthesis while cell multiplication was slightly stimulated. On the contrary, the addition of EGF to fibroblasts in a collagen lattice resulted in a stimulation of both NCP and collagen synthesis as observed in vivo. These opposite effects of EGF in a two- or three-dimensional culture system are not related to modification in number or affinity of the EGF receptors at the cell surface. These results further support the similarity in the state of differentiation of fibroblasts in a three-dimensional lattice and in vivo.