The pattern of GnRH release is associated with differential synthesis and release of LH and FSH. Using a perifusion system, we previously reported that stimulation of the LbetaT2 cell line with varying GnRH pulse frequencies resulted in differential stimulation of LHbeta and FSHbeta gene transcription, analogous to previous observations in primary gonadotropes. In the present study, we investigated the patterns of MAPK activation by GnRH and the role of MAPK in mediating the frequency-dependent effects. In static culture, ERK activation in LbetaT2 cells stimulated with continuous GnRH (10 nM) was maximal by 10 min and persisted for up to 6 h, with a return to basal levels by 20 h. In contrast, stimulation with continuous GnRH (10 nM) in perifused cells resulted in a more sustained activation of ERK. To investigate the effects of GnRH pulse frequency on ERK activation, perifused LbetaT2 cells were stimulated with pulsatile GnRH at a frequency of one pulse every 30 min or one pulse every 2 h for 20 h (10 nM, 5 min/pulse). After the final GnRH pulse, cells were lysed at frequent intervals and levels of ERK phosphorylation were measured. Under high-frequency conditions, ERK activation was maximal 10 min after the GnRH pulse and returned to baseline levels by 20 min. In contrast, under lower GnRH pulse frequency conditions, ERK activation occurred more rapidly and activation was more sustained, with a slower rate of ERK dephosphorylation. These changes resulted in different levels of nuclear phosphorylated ERK. Blockade of ERK activation abolished GnRH-dependent activation of LHbeta and FSHbeta transcription at both high and low pulse frequencies. These results demonstrate that in perifused LbetaT2 cells, distinct patterns of ERK activation/inactivation are regulated by GnRH pulse frequency, and the difference in ERK activation may be important for GnRH pulse frequency-dependent differential stimulation of LHbeta and FSHbeta gene expression.