Alterations in the frequency of calcium influx signals to rat pituitary cells can regulate the expression of gonadotropin subunit mRNAs in a differential manner, producing effects that are similar to those previously found for GnRH. The present study was conducted to investigate whether this reflects a transcriptional response to calcium pulse frequency, as determined by alterations in primary transcript (PT) expression. Perifused rat pituitary cells were given pulses of the calcium channel-activator Bay K 8644 (BK; with 10 mM KCl in the injectate) for 6 h. The response to alterations in pulse dose was examined by giving pulses of 1, 3, or 10 μM BK at 60-min intervals. Maximal increases in LHβ and FSHβ PTs were obtained with the 3-μM BK pulse dose and with the 10-μM dose for α. To investigate the effect of calcium pulse frequency, 3-μM BK pulses were given at intervals of 15, 60, or 180 min. Alpha PT was selectively stimulated by 15-min pulses and LHβ by 15- and 60-min pulses of BK. In contrast, FSHβ PT was maximally stimulated by the slower, 180-min pulse interval. These findings reveal that pulsatile increases in intracellular calcium stimulate α, LHβ, and FSHβ transcription in a differential manner. Thus, intermittent changes in intracellular calcium appear to be important in the transmission of GnRH pulse signals from the plasma membrane to the gene, and they may mediate the differential actions of pulse frequency on gonadotropin subunit gene expression.