We examined the effect of thyrotropin (TSH) stimulation of FRTL5 rat thyroid cells on ferritin H mRNA levels. On Northern blot analysis, TSH (in the presence of serum and insulin) increased ferritin H mRNA levels, with an initial response evident after 1 h of stimulation. Ferritin H mRNA levels increased approximately 4-fold over basal levels after 4 h of TSH stimulation and showed little increase thereafter, maintaining a plateau for up to 48 h of TSH stimulation. Inducers of cAMP also increased ferritin H mRNA levels in FRTL5 cells to about the same extent, but the rate of response was not as rapid as with TSH stimulation. In serum-poor medium without insulin, the TSH effect was considerably weaker, increasing only about 2-fold after 24 h of stimulation. Also in serum-poor medium, insulin-like growth factor-I alone had a weak stimulatory effect on ferritin H mRNA levels. TSH and insulin-like growth factor-I had additive effects under these conditions. Nuclear run-on transcription assays were performed using nuclei prepared from FRTL5 cells. In serum-containing medium, TSH increased the transcriptional activity of ferritin H mRNA 3-4-fold without an increase in beta-actin transcriptional activity. The kinetics of TSH stimulation of ferritin H transcriptional activity were similar to the cellular ferritin H mRNA response to TSH stimulation. Dibutyryl-cAMP (dB-cAMP) increased ferritin H transcriptional activity about 4-fold, but not as rapidly as did TSH stimulation. In summary, our data indicate that ferritin H mRNA levels in FRTL5 thyroid cells are transcriptionally regulated by both TSH and dBcAMP stimulation. These data contrast with the predominantly nontranscriptional regulation of ferritin H mRNA levels observed in other tissues. The difference in the kinetics of the response to TSH and dBcAMP is consistent with the concept that not all effects induced by TSH stimulation of thyroid cells are mediated by cAMP as a second messenger.