The mechanism of cell proliferation by a combination of thyroid-stimulating hormone (TSH) and insulin-like growth factor-I (IGF-I) was studied in rat thyroid (FRTL-5) cells. IGF-I stimulated an approximately 3.5-fold increase in the rate of Ca2+ influx sustained for at least 6 h in TSH-pretreated cells but not in quiescent cells. The significant cell proliferation was observed when TSH-primed cells were incubated with IGF-I for 24 h but not for 12 h. IGF-I stimulated the rate of Ca2+ influx in a dose-dependent manner that was similar to that for induction of DNA synthesis. Both Ca2+ influx and DNA synthesis observed in response to IGF-I in TSH-primed cells were inhibited by cobalt. In addition, the stimulations of Ca2+ influx and DNA synthesis by IGF-I were dependent on extracellular Ca2+ in TSH-pretreated cells. When TSH-primed cells were pretreated with pertussis toxin, both IGF-I-induced Ca2+ influx and DNA synthesis were abolished. However, pertussis toxin did not block the priming action of TSH or forskolin. When calcium entry was induced by Bay K8644, it stimulated cell growth in TSH-primed cells but not in quiescent cells. Moreover, cobalt and lanthanum inhibited DNA synthesis even when added several hours after the addition of Bay K8644 but not when added 24 h after the growth factor in TSH-primed cells. These findings suggest that at least two important mechanisms may work in response to IGF-I only in the TSH-primed G1 phase of the cell cycle: first, IGF-I can activate directly or indirectly the Ca2+ channel via a pertussis toxin-sensitive substrate in TSH-primed cells; and second, a long lasting calcium entry by IGF-I may be a cell cycle-dependent mitogenic signal.