5-HT4 receptors in isolated distal colon myenteric plexus of guinea-pig, mediating contraction of longitudinal smooth muscle, have been further characterized by selective agonists and antagonists. The indole agonists, 5-HT and 5-methoxytryptamine (5-MeOT), were full agonists (relative to 5-HT) with potency values (pEC50) of 8.0 +/- 0.1 (n = 50) and 7.8 +/- 0.1 (n = 12), respectively. 5-HT4 receptor agonists of other structural classes, including benzimidazolones (BIMU 1 and BIMU 8), and benzamides ((S)-zacopride, (R)-zacopride, renzapride, SC 49518) were partial agonists with intrinsic activities less than that of 5-HT. In general, the potencies for these compounds at 5-HT4 receptors in guinea-pig colon were similar to the potencies seen in the rat isolated oesophagus, where 5-HT4 receptors mediate relaxation. GR 113808 ¿[1-[2-[(methylsulfonyl)amino]ethyl]-4-piperidinyl] methyl1-methyl-1H-indole-3-carboxylate¿, RS 39604 ¿1-[4-amino-5-chloro-2-(3, 5-dimethoxybenzyloxy)phenyl]-3[1-[2-[(methylsulfonyl)amino] ethyl]-4-piperidinyl]-1-propanone hydrochloride and SB 204070 ¿(1-n-butyl-4-piperidinyl)methyl 8-amino-7-chloro-1, 4-benzodioxane-5-carboxylate¿ antagonized 5-HT responses with pA2 values of 9.1 +/- 0.1, 9.0 +/- 0.2 and 11.0 +/- 0.1, respectively. These affinity values were similar to those obtained at 5-HT4 receptors in isolated rat oesophagus (9.0+/- 0.4, 9.3 +/- 0.1 and 10.6 +/- 0.1 respectively). Despite these operational similarities between 5-HT4 receptors in guinea-pig colon and rat oesophagus, several novel compounds have revealed important differences between 5-HT4 receptors in the two tissues. For example, the substituted benzoate, RS 23597 ¿3-(piperidine-1-yl) propyl-4-amino-5-chloro-2-methoxybenzoate hydrochloride, acted as a partial agonist (intrinsic activity 0.5) in guinea-pig colon with a potency of 7.6 +/-0.1 (n = 16). In isolated rat oesophagus, however, this compound was a surmountable antagonist (pA2 = 7.8 +/- 0.1) with no intrinsic activity. In contrast, the substituted naphthalimide (S)RS 56532 ¿(S)-6-amino-5-chloro-2-(1-azabicyclo[2, 2, 2]octan-3-yl) 2,3-dihydro-1H-benz[de] isoquinoline-1,3-dione hydrochloride¿, was a potent (pEC50 = 7.9 +/- 0.1), efficacious partial agonist (intrinsic activity = 0.8) in the rat oesophagus. However, in guinea-pig colon, it was a surmountable antagonist with an affinity (pKB) of 9.4 +/- 0.1. Furthermore, several novel, selective, 5-HT4 compounds also showed opposing patterns of intrinsic activities similar to those described for RS 23597 and (S)RS 56532. It is concluded that these differences are inconsistent with differences in 5-HT4 receptor reserves, and may suggest that 5-HT4 receptors in the guinea-pig colon and the rat oesophagus can be operationally distinguished.