The intracellular pathways mediating GnRH regulation of gonadotropin subunit transcription remain to be fully characterized, and the present study examined whether calcium/calmodulin-dependent kinase II (Ca/CAMK II) plays a role in the rat pituitary. Preliminary studies demonstrated that a single pulse of GnRH given to adult rats stimulated a transient 2.5 fold rise in Ca/CAMK II activity (as determined by an increase in Ca/CAMK II phosphorylation), with peak values at 5 min, returning to basal 45 min after the pulse. Further studies examined the , LH and FSH transcriptional responses to GnRH or Bay K 8644+KCl (BK+KCl) pulses in vitro in the absence or presence of the Ca/CAMK II-specific inhibitor, KN-93. Gonadotropin subunit transcription was assessed by measuring primary transcripts (PT) by quantitative RT-PCR. In timecourse studies both GnRH and BK+KCl pulses given alone increased all 3 subunit PTs after 6 h (2- to 4-fold). PT responses to GnRH increased over time (3- to 8-fold over basal at 24 h), while BK+KCl was ineffective after 24 h. KN-93 reduced the LH and FSH transcriptional responses to GnRH by 50-60%, and completely suppressed the PT response. In contrast, KN-93 showed no inhibitory effects on basal transcriptional activity or on LH or FSH secretion. In fact, KN-93 tended to increase both basal , LH and FSH PT levels, and enhance LH secretory responses to GnRH. These results reveal that Ca/CAMK II plays a central role in the transmission of pulsatile GnRH signals from the plasma membrane to the rat , LH and FSH subunit genes.