Although cells of the immune system can produce thyroid-stimulating hormone (TSH), the significance of that remains unclear. Using 5' rapid amplification of cDNA ends (RACE), we show that mouse bone marrow (BM) cells produce a novel in-frame TSHbeta splice variant generated from a portion of intron 4 with all of the coding region of exon 5, but none of exon 4. The TSHbeta splice variant gene was expressed at low levels in the pituitary, but at high levels in the BM and the thyroid, and the protein was secreted from transfected Chinese hamster ovary (CHO) cells. Immunoprecipitation identified an 8 kDa product in lysates of CHO cells transfected with the novel TSHbeta construct, and a 17 kDa product in lysates of CHO cells transfected with the native TSHbeta construct. The splice variant TSHbeta protein elicited a cAMP response from FRTL-5 thyroid follicular cells and a mouse alveolar macrophage (AM) cell line. Expression of the TSHbeta splice variant, but not the native form of TSHbeta, was significantly upregulated in the thyroid during systemic virus infection. These studies characterize the first functional splice variant of TSHbeta, which may contribute to the metabolic regulation during immunological stress, and may offer a new perspective for understanding autoimmune thyroiditis.