Substance P (SP) has been suggested to regulate gonadotroph function both directly and indirectly in different species. In pigs, the possible role of hypothalamic and pituitary SP in the control of LH release has not been examined. Here, we investigated SP effects on basal and GnRH-stimulated LH secretion by porcine gonadotrophs in vitro, in both static and dynamic culture systems. SP concentrations of 100 nM or above stimulated LH release from monolayer cultures without affecting intracellular LH content. In the same cultures, SP potentiated GnRH (10 nM)-stimulated LH release and reversed the GnRH-induced decrease of gonadotroph LH stores. The GnRH, but not the SP, effect was completely blocked by the potent GnRH-receptor antagonist antide. In superfused pituitary fragments, three successive pulses of SP or GnRH also stimulated LH release, yet the combined administration of both factors did not result in a synergistic stimulation. These results demonstrate that SP acts directly on porcine gonadotrophs to stimulate LH release, and to maintain the levels of hormonal stores, through a GnRH receptor-independent mechanism. Furthermore, our findings suggest that continuous exposure of gonadotrophs to SP would potentiate GnRH-stimulated LH secretion, thus supporting a possible role of SP as modulator of porcine gonadotroph function.