Follicular development and ovulation are suppressed during lactation in various mammalian species, mainly due to the suppression of pulsatile gonadotropin-releasing hormone (GnRH)/luteinizing hormone (LH) secretion. Metastin (Kisspeptin-54), a KiSS-1 gene product, is an endogenous ligand for GPR54, a G-protein-coupled receptor, and suggested to play a critical role in regulating the gonadal axis. The present study, therefore, aims to determine whether metastin (kisspeptin-54)-GPR54 signaling in discrete brain areas is inhibited by the suckling stimulus that causes suppression of LH secretion in lactating rats. Quantitative RT-PCR revealed that the KiSS-1 mRNA level was significantly lower in the arcuate nucleus-median eminence (ARC-ME) region in lactating ovariectomized (OVX) and estrogen-treated OVX rats than in non-lactating controls. KiSS-1 mRNA in the anteroventral periventricular nucleus (AVPV) was kept at a low level in both lactating and non-lactating rats despite estrogen treatment. GPR54 mRNA levels were significantly lower in lactating than non-lactating rats in the AVPV, but the levels in lactating mothers of the preoptic area (POA) and ARC-ME were comparable to non-lactating controls. Although KiSS-1 mRNA-expressing cells or metastin (kisspeptin-54)-immunoreactivities were densely located in the ARC of non-lactating controls, few were found in the ARC of lactating OVX animals. Various doses of metastin (Kisspeptin-54) (0.02, 0.2 and 2 nmol) injected into the third ventricle caused a significant increase in LH secretion in both lactating and non-lactating OVX rats, suggesting that lactating rats are responsive to metastin (kisspeptin-54) stimulus. Thus, the present study demonstrated that KiSS-1 mRNA/metastin (kisspeptin-54) expression is inhibited in the ARC by the suckling stimulus, suggesting that the inhibition is most probably involved in suppressing LH secretion in lactating rats.