Abstract IL-18 is a pleiotropic cytokine also proposed to have a role in modulating immune function during stress. Initially found in immune cells, IL-18 mRNA is detectable in several tissues including the cells of the zona reticularis and the zona fasciculata of the adrenal cortex, where its levels are elevated by acute stress or adrenocorticotropic hormone treatment. In the present study, we compared the expression of IL-18 in the adrenal cortex with that of spleen and duodenum, two other IL-18-positive tissues. In situ hybridization showed that, in contrast to the adrenal cortex, in spleen and duodenum IL-18 is primarily localized to immune cells. In duodenum, IL-18 mRNA was also detectable in epithelial cells. Northern blot demonstrated that while the adrenal gland synthesized IL-18 mRNA of 1.1 kb, spleen and duodenum produced a 0.9-kb transcript. RT-PCR, sequencing, Western blot, primer extension, and rapid amplification of cDNA end analysis demonstrated that the three tissues synthesize IL-18 mRNAs containing the same coding region and produce the same IL-18 peptide, but differ in the length of their 5′-untranslated region, indicating tissue-specific usage of the promoter region. Finally, in contrast to the adrenal gland, adrenocorticotropic hormone treatment did not increase the levels of IL-18 mRNA in spleen and duodenum. These results demonstrate tissue-specific expression and promoter usage of IL-18 gene and suggest that the adrenal cortex and not immune cells may be the source of IL-18 during stress.