We previously have demonstrated that rat adipose tissue expresses TSH receptor (TSHR) messenger RNAs (mRNAs) at levels approaching those detected in the thyroid. Furthermore, we recently reported that TSHR mRNA is detected in fibroblast-like 3T3-L1 cells after their hormone-induced differentiation into adipocytes. TSH induces cAMP formation and lipolysis in differentiated 3T3-L1 cells. We now show that, in Northern blot analyses, TSH-induced down-regulation of TSHR mRNA levels, which can be duplicated by forskolin and dibutylyl cAMP, i.e. which is cAMP-mediated. We also have demonstrated that a beta-adrenergic stimulant, which stimulates cAMP formation in adipocytes, induces a down-regulation of TSHR mRNA levels in 3T3-L1 adipocytes. Nuclear run-on assays show that the ability of TSH/cAMP to decrease TSHR mRNA levels in 3T3-L1 cells reflects transcriptional regulation. This report also demonstrates that TSHR gene expression in 3T3-L1 adipocytes is regulated in a manner distinct from that observed in thyroid cells. Thus, in fully differentiated 3T3-L1 adipocytes, TSH-induced down-regulation of TSHR mRNA levels is evident within 1 h and is near maximum within 4 h after addition of TSH. A transient increase of TSHR gene expression, which has been demonstrated in FRTL-5 thyroid cells, was not observed in 3T3-L1 adipocytes. The down-regulation of TSHR gene expression induced by TSH/cAMP in 3T3-L1 cells is cycloheximide-insensitive, suggesting that continuous protein synthesis is not required for this process. In contrast, the down-regulation of TSHR gene expression observed in FRTL-5 cells is sensitive to cycloheximide. In both FRTL-5 thyroid cells and 3T3-L1 adipocytes, insulin or serum increased TSHR mRNA levels. Although insulin or serum was required for the TSH-induced down-regulation of TSHR mRNA levels in FRTL-5 thyroid cells, neither insulin nor serum was required for TSHR down-regulation in 3T3-L1 adipocytes. These findings demonstrate that TSH/cAMP regulates TSHR mRNA levels in adipocytes via a regulatory system distinct from that used in FRTL-5 cells. This report further demonstrates that adipose cells do not express thyroid transcription factor-1, which interacts with the TSHR promoter region in FRTL-5 cells, and that 3T3-L1 nuclear extracts exhibit a different binding activity to the cAMP-response element-like element in the TSHR promoter region compared with extracts from FRTL-5 cells.