TNF-alpha is initially synthesized as a membrane-anchored precursor protein and processed proteolytically by a matrix metalloproteinase (MMP)-like enzyme. In order to establish an efficient expression system of TNF-alpha in mammalian cells without involvement of the extracellular enzyme, an expression plasmid (pCN-alb-TNF) was constructed with a signal sequence of the rat albumin gene as a module for secretion. The highest level of production of TNF-alpha was observed in the clone CT-3 by SDS-PAGE and Western blot analysis. Biological activity of the secretion was revealed by repression of catalase gene expression in hepatoma cells and cytotoxity to L929 cells. Attachment of the signal peptide to mature form resulted in the enhancement of production of the cytokine.