In FRTL-5 cells, we and others have shown that TSH and insulin-like growth factor-I (IGF-I) stimulate DNA synthesis synergistically. The present study was undertaken to determine whether interaction between TSH and IGF-I also affects protein synthesis in this cell line, and if so by what mechanism. Quiescent cells were treated with TSH and/or IGF-I and [3H]valine incorporation into the acid-insoluble fraction was measured as a parameter of protein synthesis. Similar to their effects on cell proliferation, TSH or IGF-I alone induced protein synthesis only slightly, but treatment with a combination of TSH and IGF-I (or insulin with about a 100-fold higher concentration than IGF-I) greatly increased protein synthesis. The presence of IGF-I potentiated a TSH-concentration-dependent increase in protein synthesis and in DNA synthesis. In addition, we observed this potentiation when the cells were treated with other cAMP-generating agents and cAMP analogues instead of TSH. We have shown that priming with TSH potentiated DNA synthesis induced by IGF-I, whereas pretreatment with IGF-I enhanced protein synthesis induced by TSH. This observation suggested that protein synthesis and DNA synthesis were potentiated through different mechanisms. From an analysis of cAMP production, it appears that the potentiation of protein synthesis may be explained by an IGF-I-dependent increase in cAMP production induced by TSH at least in part. On the other hand, IGF-I and TSH stimulated (alpha-aminoisobutyric acid (AIB) uptake synergistically, but RNA synthesis induced by IGF-I was depressed by TSH. From these results, we concluded that in FRTL-5 cells, IGF-I potentiated protein synthesis induced by TSH by means of complex mechanisms and the interaction between IGF-I and cAMP-dependent pathways may also have a physiological meaning in regulating protein anabolism.