Neuropeptide FF (NPFF) and NPVF, two closely NPFF related peptides, have different affinities for the two NPFF receptors (NPFF1 and NPFF2). To assess the peripheral effects of NPFF receptors in the gastrointestinal tract motility, NPFF and NPVF were tested in the mouse isolated distal colon. Both NPFF (1-15 microM) and NPVF (1-15 microM) dose-dependently caused significant colonic contractions. Pre-treatment with the putative NPFF antagonist, BIBP3226 (30 microM) abolished the contractile responses to the two neuropeptides (3 microM). They had no additional contractile activities in colonic preparations contracted by Nomega-nitro-L-arginine (30 microM). Moreover, the contractions of these two neuropeptides were weakened by L-arginine (2 mM). The responses to NPFF (5 microM) and NPVF (5 microM) were not modified by atropine or naloxone (1 microM). Furthermore, NPFF (1 microM) and NPVF (1 microM) did not influence the contractive responses to acetylcholine (0.1-10 microM), morphine (1 microM) or nociceptin (0.1 microM). These data suggest that NPFF and NPVF cause contractions of the mouse distal colon via their NPFF receptors and this effect is mediated by NO but not by cholinergic pathways, independently from opioid system. In addition, the isolated bioassay may be applied as a simple parameter to characterize the potential NPFF agonists and antagonists.