1. In the presence of atropine, mepyramine and ranitidine, electric field stimulation of the guinea-pig isolated ileum longitudinal muscle-myenteric plexus preparation resulted in a two component non-adrenergic non-cholinergic contraction. The initial contraction had a duration of approximately 1 s whereas the second contraction lasted approximately 10 s. The second contraction was completely inhibited by tetrodotoxin (0.2 x 10(-6) M) with minimal effect on the initial contraction. Phentolamine (3 x 10(-6) M), propranolol (3 x 10(-6) M) and hexamethonium (10(-4) M), did not significantly reduce either component of the contractile response. 2. The neurokinin NK1 receptor antagonists, GR82334 and GR71251, produced concentration-related (EC50 = 564 and 173 nM respectively) inhibitions of the second contraction with no effect on the initial contraction. The neurokinin NK2 receptor antagonists MEN 10207 and Ac-Leu-Asp-Gln-Trp-Phe-Gly-NH2 (R 396), 1 x 10(-9)-10(-5) M, were without effect on either component of the contractile response. 3. Concentration-related inhibitions of the second contraction, with no effect on the initial contraction, were observed after inclusion of the histamine H3 receptor agonists (R)-alpha-methylhistamine (pD2 = 7.6), N alpha-methylhistamine (pD2 = 7.7) and N alpha,N alpha-dimethylhistamine (pD2 = 6.3). Histamine also inhibited the second contraction (pD2 = 6.2) in a concentration-related manner but produced a lower maximum inhibitory effect than the other agonists tested. 4. Inclusion of the H3 receptor antagonists, thioperamide, burimamide, impromidine and phenylbutanoylhistamine, caused parallel concentration-related rightward shifts in the concentration-response curve to (R)-alpha-methylhistamine. In each case, Schild analysis of these data gave slopes not significantly different from unity. Antagonist affinity values for thioperamide (pA2 = 8.2), burimamide (pA2 = 7.0) and impromidine (pA2 = 7.0) were consistent with values obtained in other assays of the H3 receptor. However, phenylbutanoylhistamine (pA2 = 5.8) and betahistine (pKB < 4) had affinities more than ten fold lower than values obtained in other assays of the H3 receptor.5. Exposure of the tissues to N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (10-6 M) for 7-30min followed by extensive washing, had no effect on basal contractions, but produced a rightward shift in the concentration-response curves to (R-alpha-methylhistamine, Nalpha"-methylhistamine, Nalpha",Nalpha-dimethylhistamine and histamine. This treatment also resulted in a decrease in the maximum inhibitory response obtainable. Apparent agonist affinity (pKD) values of 7.01, 7.06, 6.09 and 6.13 were estimated for (R)-alpha-methylhistamine, Nalpha-methylhistamine, Nalpha',Nalpha"-dimethylhistamine and histamine respectively.6. In conclusion, pharmacological analysis has revealed that histamine H3 receptors in the guinea-pig ileum modulate the release of non-adrenergic non-cholinergic neurotransmitters, one of which is probably substance P. In addition we have identified N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline as an irreversible antagonist at H3 receptors and have used this compound to estimate apparent affinity values of agonists at H3 receptors in this preparation.