To investigate the regulation of expression of the TSH receptor (TSHR) in extrathyroidal tissues, the level of TSHR messenger RNA (mRNA) and TSH-dependent signal transduction were determined in isolated rat adipocytes and cultured preadipocytes. The epididymal, sc, and perirenal, but not the interscapular brown adipose tissues, possessed TSHR mRNA and increased cAMP responses to TSH and were thus used as the source of preadipocytes. Morphological analysis revealed that the combination of insulin and T3 most effectively caused the differentiation of rat preadipocytes. These differentiated preadipocytes exhibited increased cAMP production in response to TSH. The addition of FCS to the culture medium inhibited the differentiation of rat preadipocytes as well as TSH-stimulated production of cAMP. The stimulation of differentiation was associated with an increased expression of TSHR mRNA levels, whereas the inhibition of differentiation was associated with a decreased expression of TSHR mRNA, as detected by Northern blot analysis. The results indicate that the expression and function of the TSHR in cultured rat preadipocytes are closely related to cellular differentiation. Cultured rat preadipocytes appear to provide a useful system for studying the mechanism of extrathyroidal expression of TSHR.