TNF-stimulated gene (TSG)-14 was originally identified as a TNF-inducible gene in a differentially screened cDNA library derived from TNF-treated normal human FS-4 fibroblasts. Analysis of the TSG-14 cDNA sequence revealed a major open reading frame encoding a protein of 381 amino acids, including a hydrophobic signal peptide sequence. The predicted protein shows 23 to 27% sequence homology to C-reactive protein and serum amyloid P-component, members of the pentaxin family of acute phase proteins. In addition, TSG-14 protein contains a sequence motif common among the pentaxin proteins. The ability of the TSG-14 cDNA to encode a protein of the correct molecular size was confirmed in a cell-free transcription/translation system. In vitro translation in the presence of microsomes confirmed that the protein has a cleavable signal peptide sequence, and that it is glycosylated. TSG-14 mRNA is rapidly elevated from almost undetectable levels in untreated FS-4 cells to high levels in cells treated with TNF or IL-1. A moderate increase in TSG-14 mRNA was observed in FS-4 cells treated with the glucocorticoid dexamethasone. Nuclear run-on analysis indicated that TNF induces the expression of the TSG-14 gene at the transcriptional level, and that de novo protein synthesis is not required for induction of TSG-14 mRNA. Expression of TSG-14 mRNA was also detected after exposure to TNF in vascular endothelial cells; however, little or not expression of TSG-14 message was observed in cell lines derived from malignant tumors. Our data strongly suggest that TSG-14 is a novel member of the pentaxin family of acute phase proteins.