We have isolated an approximately 22-kb TNF locus (encoding tumor necrosis factor) from a rat genomic library and sequenced the 7105-bp fragment that comprises the TNF-α and TNF-β genes, including their flanking sequences. The two genes are tandemly arranged with TNF-β 5' to TNF-α and separated by approximately 1.1 kb of intergenic space, and each gene consists of four exons and three introns, similar to those of the other species examined thus far. Comparison analysis showed that the rat TNF have high sequence homology with the mouse TNF (TNF-α, 86.5%; TNF-β, 89.3%) and relatively low homology with the human, rabbit, and porcine TNF. The upstream sequence of rat TNF-α. contains a number of sequence motifs implicated in the expression and regulation of eukaryotic genes, including binding sites for the transcription factors Sp-1, Ap-2, IFN.l and NF-KB. The possible significance of potential regulatory sequence elements found in the rat TNF-α in the context of transcriptional regulatory mechanisms is discussed. © 1993.