The effects of trypsin and arginine analogues, alone or in combination, on half-maximal non-adrenergic, non-cholinergic (NANC) relaxation elicited by different pulse trains of electrical field stimulation were studied in the rat gastric fundus in order to investigate further the relative contribution of peptides and NO. Trypsin (1 microM) partially inhibited electrically-induced NANC relaxation especially when longer pulse trains were used. L-NOARG, L-NAME and L-NMMA, but not D-NOARG or D-NAME (3-300 microM) produced concentration-dependent inhibition of the electrically induced NANC relaxation. L-Arginine (L-Arg), but not D-Arginine (D-Arg) (3.8 microM-3.8 mM) produced a concentration-dependent reversal of the inhibitory effect of L-NOARG IC50 (38 microM). Neither L-NOARG (38 microM) nor L-Arg (380 microM) influence submaximal relaxation induced by VIP (3 nM), isopropylnoradrenaline (10 nM), ATP (10 microM) or sodium nitroprusside (300 nM). Moreover L-NOARG (100 microM) did not influence neurally-induced VIP release. L-NOARG inhibition of NANC relaxation was significant only when short pulse trains were used, while trypsin showed significant inhibition only of relaxation induced by longer pulse trains. These results suggest that the relaxation induced by the activation of the NANC inhibitory neurotransmission of the rat gastric fundus consists of at least two components, one trypsin-sensitive and the other trypsin-resistant, to which VIP and NO contribute, respectively.