TSH-releasing hormone (TRH) binds to a membrane receptor that activates several intracellular signaling pathways and increase transcription of the TSH (TSH) and prolactin (PRL) genes. Although TRH induces TSH and PRL gene expression, the underlying mechanism is not clear. In this report, we examined the role of the Ca²⁺/calmodulin-dependent protein (CaM) kinases cascade in mediating TRH stimulated transcription of TSH and PRL. RT-PCR and Western blot analysis were used to show that CaM-kinase kinase (CaM-KK) and CaM-kinase IV (CaM-KIV) were present in rat anterior pituitary and its cell line GH3. Next the effects of constitutively active CaM-KIV (CaM-KIVc) or its dominant negative mutant (CaM-KIVdn) on TSH and PRL promoter activity were tested in GH3 cells. Results showed that either CaM-KIVc alone or an upstream kinase, CaM-KK induced the activity of both TSH and PRL promoters. Exposure of the GH3 cells to 100 µM TRH induced CaM-KIV activity within 5 min and as expected also increased both TSH and PRL promoter activity. In contrast, cells carrying the CaM-KIVdn isoform had suppressed TRH-induction of both TSH and PRL promoter activity. These results indicate that CaM-KK-CaM-KIV cascade likely play an important role in TRH-induction of TSH and PRL transcriptional activity in pituitary cells.