Toll-like receptors (TLRs) are essential components of the innate immune system. TLR5 is the receptor for flagellin, the principal protein component of bacterial flagella. The TLR5 gene has 6 exons. In an RT-PCR analysis, we found long TLR5 transcripts, in addition to those of the expected size (short TLR5 transcripts). A sequence analysis revealed that the long TLR5 transcripts contain a new exon of 94 nucleotides located between previously reported exons IV and V in the 5' untranslated region (5' UTR). A real-time PCR analysis of the two alternatively spliced variants in various cell lines showed that the long TLR5 transcripts are abundantly expressed in nonimmune cells. The ratios of long/short transcripts in human nonimmune cell lines, such as A549, T98G, HaCaT, H460, HEK-293, and Caco-2 cells, and primary mesenchymal stem cells were in the range of 1.25 to 4.31. In contrast, those of human monocytic THP-1 and U937 cells and E6.1 T cells and Ramos B cells were around 0.9. These ratios in human monocytic THP-1 cells were decreased by treatment with IFN-γ in a concentration-dependent manner. Based on our findings, we suggest that the newly found long TLR5 transcripts may be involved in the negative regulation of TLR5 expression and function.