The number of pituitary cells, their size, hormonal content and release and response to external cues varies between day and night and during the estrus cycle. Previous studies have demonstrated that pituitary cells proliferate rhythmically and that estradiol (E2) is a mitogen of αT3 cells. We, therefore, studied the effect of gonadotropin releasing hormone (GnRH) and E2, on the cell cycle in primary cultures of mouse pituitary cells and in the gonadotroph cell line LβT2. We found that GnRH and E2 modulate the cell cycle in a time dependent manner and induce proliferation in cultures of mouse pituitary and LβT2 cells. GnRH induces proliferation in cells isolated in the morning of the estrus day and increases the number of cells in G2 stage when isolated in noon and evening. However, the transition into the G1 stage is enabled only by co-addition of E2 and GnRH. GnRH stimulates LH release from LβT2 cells after 2 days via exocytosis while after 4 days in culture, the increase in LH release may be accounted for by the increase in cell number. E2 enhanced the GnRH response after 2 days, and abolished it after 4 days in culture. Furthermore, E2 has no effect on LH release and cell number after 2 days in culture, however, after 4 days in culture, E2 had no effect on the total amount of LH released but inhibited LH release per cell due to increase in cell number. Our results show that GnRH and E2 function to shorten the cell cycle and regulate the cell number of each stage of the cell cycle. The effect of GnRH and E2 on the cell cycle is dependent on the circadian time. This mechanism may serve to modulate the size and function of the pituitary cell population and consequently the function of pituitary gonadotrophs regulating the surge of LH release before ovulation.