We investigated whether JNK and p38, mediate gonadotropin subunit transcriptional responses to pulsatile GnRH in normal rat pituitaries. A single pulse of GnRH or vehicle was given to female rats in vivo, pituitaries collected, and phosphorylated JNK and p38 measured. GnRH stimulated an increase in JNK phosphorylation within 5 min, which peaked 15 min post-GnRH (3 fold). GnRH also increased p38 phosphorylation 2.3 fold, 15 min post-stimulus. Rat pituitary cells were given 60 min pulses of GnRH or media plus the JNK inhibitor SP600125 (SP, 20uM), p38 inhibitor SB203580 (SB, 20uM) or vehicle. In vehicle-treated groups, GnRH pulses increased LH and FSH primary transcript (PT) levels 3 fold. SP suppressed both basal and GnRH-induced increases in FSH PT by half, but the magnitude of responses to GnRH was unchanged. In contrast, SP had no effect on basal LH PT, but suppressed the stimulatory response to GnRH. SB had no effect on the actions of GnRH on either LH or FSH PTs. L-T2 cells were transfected with dominant/negative (DN) expression vectors for MKK-4 and/or MKK-7 plus a rat LH promoter-luciferase construct. GnRH stimulated a 50-fold increase in LH promoter activity, and the combination of MKK-4 and 7 DNs suppressed the response by 80%. Thus, JNK (but not p38) regulates both LH and FSH transcription in a differential manner. For LH, JNK is essential in mediating responses to pulsatile GnRH. JNK also regulates FSH transcription (i.e. maintaining basal expression), but does not play a role in responses to GnRH.