We have investigated the mechanism by which TSH pretreatment potentiates insulin-like growth factor I (IGF-I)-induced DNA synthesis in FRTL-5 cells. As previously described, pretreatment with TSH increased IGF-I-induced DNA synthesis, suggesting that the effect of TSH is mediated through the cAMP pathway. TSH and A kinase activators required at least 12 h to precondition cells to respond to IGF-I stimulation. The presence of cycloheximide abolished the effect of TSH to increase IGF-I-induced DNA synthesis. When the time course of thymidine uptake after IGF-I addition was studied, TSH pretreatment increased the maximum DNA incorporation and shortened the G1 phase interval. These results indicated that some proteins induced by TSH are required for the effect of TSH on IGF-I activity, and the proteins are important for cell cycle progression. Cyclins are key regulators of the cell cycle; therefore, we investigated the expression of cyclins D1 and E after TSH stimulation. TSH- and A kinase-activating agents increased the expression of cyclins D1 and E after 24 h. The same amounts of cyclins D1 and E induced by IGF-I were increased after TSH pretreatment. TSH pretreatment induced the expression of G1 cyclin in FRTL-5 cells, and IGF-I caused the accumulation of enough G1 cyclins to drive the cell cycle from G1 to S phase in a short time, which accounts for the effect of TSH on IGF-I induced DNA synthesis.