The TSH receptor is a G-protein-coupled seven transmembrane segment receptor. The interaction between TSH and its receptor mediates signal transduction by activating adenylyl cyclase through Gsα. There are four forms of Gsα (two short [45 kDa] and two large [52 kDa]), arising from alternative splicing of exon 3 of the Gsα gene. Gsα-1 and -2 contain exon 3, whereas exon 3 is spliced out in Gsα-3 and -4. The inclusion of a serine residue at the 3' splice junction of exon 3 distinguishes Gsα-2 and -4 from Gsα-1 and -3. The expression of different Gsα forms appears to be tissue-specific. In this study, we have examined the Gsα splice variants in 26 human thyroid tumor specimens and rat thyroid tissues as well as a rat FRTL-5 cell line. Furthermore, we have studied the regulation of the Gsα gene expression by TSH and cAMP in FRTL-5 cells. We found that Gsα-1 and -4 mRNA were present in both human and rat thyroid cells, although Gsα-4 was more abundant in human thyroid cells as compared to rat thyroid and FRTL-5 cells. The Gsα mRNA can be easily amplified by RT-PCR regardless of tumor type and stage, suggesting that Gsα gene expression in thyroid tumors may not be markedly affected by dedifferentiation of thyroid cells.Both TSH and 8-bromo-cAMP, a cAMP analog, can stimulate the Gsα gene expression in FRTL-5 cells with maximal effect by 6 h and 1 h, respectively. The addition of cycloheximide to the culture of FRTL-5 cells abolished the effect of bTSH, but not that of 8-bromo-cAMP, on the expression of the Gsα gene. Cellular cAMP measurements showed that bTSH-stimulated cAMP production was significantly reduced to the basal level after addition of cycloheximide. These results suggest that regulation of the Gsα gene expression by TSH is mediated by a cAMP-dependent process and requires new protein synthesis.