The extracellular matrix is composed of a large number of different modular proteins. Matrilin-2 is a newly described member of the protein superfamily with von Willebrand factor A-like modules. To examine the expression of matrilin-2 in human skin, the distribution of protein and mRNA was studied by immunohistochemistry and in situ hybridization. In addition, immunoblotting and real-time reverse transcription polymerase chain reaction were used to investigate the expression of matrilin-2 in keratinocyte and fibroblast cultures. In vivo, keratinocytes and fibroblasts were both found to express matrilin-2 mRNA and deposit the protein at the basal side of the dermal-epidermal basement membrane. Matrilin-2 molecules synthesized by the two cell types in vitro appeared to be processed differently by cell-associated proteases. Transcription of matrilin-2 mRNA in keratinocytes was enhanced by a diffusible factor produced by fibroblasts, suggesting a regulatory mechanism for the production of extracellular matrix at the dermal-epidermal junction. These findings demonstrate that matrilin-2 is expressed in normal skin by keratinocytes and fibroblasts and may thus contribute to cutaneous homeostasis.