A second TNF gene (TNF2) has been cloned and sequenced in rainbow trout. In common with the first TNF gene isolated (TNF1), this gene is more TNFalpha-like than TNFbeta-like. The full length cDNA is 1519 bp, containing a 765 bp open reading frame. The gene has four exons, of 380,49,60 and 1030 bp, respectively. Analysis of the 5' flanking regions of TNF1 and TNF2 reveals several interesting differences in identified transcriptional regulatory elements, with a CATAAA box present 26 bp upstream of the transcription start in both genes. Expression analysis in LPS stimulated macrophages has shown a much stronger expression of TNF2 relative to TNF1, with expression being detected earlier and lasting longer. (C) 2001 Elsevier Science Ltd. All rights reserved.