Medullary thick ascending limb of Henle's loop (mTALH) tubules, isolated from kidneys of male Sprague-Dawley rats, expressed the gene for tumor necrosis factor (TNF) and released this cytokine when challenged with lipopolysaccharide (LPS). The TNF produced was biologically active, as determined by cytotoxic activity present in supernatants from LPS-stimulated mTALH, using the TNF-sensitive murine fibrosarcoma cell line, WEHI 164. The amount of TNF produced, approximately 75 nM, has previously been shown to affect ion transport in the mTALH. The TNF-mediated cytotoxicity (and ion transport effects) were completely neutralized with a polyclonal anti-TNF antisera. Further, immunoprecipitation experiments demonstrated that the 17 kDa TNF monomer was formed by de novo protein synthesis. In contrast, the mTALH did not produce the related cytokine, lymphotoxin (LT). Production of TNF was confirmed by demonstrating the accumulation of a 1.6 kb TNF mRNA by Northern blot analysis; mRNA for LT was not detected. Expression of the TNF gene in the mTALH was confirmed by the polymerase chain reaction (PCR). Southern blot analysis and ethidium bromide staining of the resultant PCR products revealed the expected 276 bp sequence of TNF DNA for the mTALH. We have demonstrated that mTALH tubules stimulated with LPS express the gene for TNF, but not LT, and release biologically active TNF. TNF is an important mediator of septic shock and may contribute to changes in renal function associated with endotoxemia. Production of TNF by the mTALH may be an important autocrine regulatory mechanism for this nephron segment.