The neuropeptide PACAP regulates numerous endocrine functions such as growth, metabolism and reproduction. In pituitary gonadotrophs, PACAP acts as a potent stimulator of gonadotrophin gene transcription and secretion, and potentiates the stimulatory effects of GnRH. We have examined the combinatorial effects of PACAP with GnRH or other activators of GnRH-signalling pathways in mouse αT3-1 gonadotroph and rat PC12 phaeochromocytoma cells. αT3-1 cells transiently transfected with the human glycoprotein hormone α-subunit promoter (αGSU-LUC) were potently stimulated by both GnRH and PACAP (both 100 nM) by 31.0±5.8 and 29.2±4.5-fold, respectively (P<0.05). However, combined GnRH and PACAP treatment caused a synergistic increase in promoter activity (169.4±6.1-fold, P<0.001). To examine whether this synergism was apparent in other PACAP target cells, PC12 cells were transiently transfected with a range of reporter constructs (PACAP-LUC, Gal4-Elk-1, Gal4-CREB, Gal4-SF-1, Gal4-LRH-1) and subsequently stimulated with 100 nM PACAP, 56 mM KCl (a depolarising concentration mimicking the calcium component of the GnRH pathway) or combination of both. Although PACAP and KCl individually stimulated the PACAP (7.6±0.2 and 2.4±0.2), Elk-1 (10.9±0.4 and 2.3±0.2) and CREB (4.5±0.1 and 2.6±0.1) reporter constructs, no synergistic effect was observed. Surprisingly, neither PACAP nor KCl altered the SF-1 or LRH-1 reporters alone, but did so in combination (by 2.9- and 1.8-fold, respectively). These data suggest that PACAP can cause synergistic stimulation of specific target genes, in combination with either GnRH or increased calcium influx. This effect is evident on the αGSU promoter, a known SF-1 target gene. As SF-1 and LRH-1 activity was similarly synergistically affected, it is possible that these nuclear receptors mediate the combined effects of GnRH and PACAP in pituitary gonadotrophs.