1. Prostaglandin E2 (PGE2) relaxes circular smooth muscle of the rabbit isolated jugular vein at very low concentrations (mean pIC50 against histamine-induced contraction = 9.34). This effect is not blocked by the EP1-receptor antagonist, AH 6809 (2 microM). 2. From a group of prostaglandin E analogues examined, 16,16-dimethyl PGE2, misoprostol, 11-deoxy PGE2-1-alcohol and 11-deoxy PGE1 were highly potent relaxant agents, whereas 17-phenyl-omega-trinor PGE2, MB 28767 and butaprost had low potency and sulprostone and oxoprostol were virtually inactive. 3. Comparison of the jugular vein data with published data for inhibitory agonist potencies on the cat trachea (EP2 preparation) and the field-stimulated guinea-pig vas deferens (EP3) indicates that the EP-receptor in the rabbit jugular vein is closest to the EP2 subtype. However, the correlation is not entirely convincing. For example, butaprost, 16,16-dimethyl PGE2 and 11-deoxy PGE1 are of similar potency on the cat trachea, whereas butaprost is about 300 times less potent than the other two analogues on the jugular vein. The existence of more than one EP2-receptor appears possible. 4. It was felt that the activity of butaprost required further investigation in view of the claim that it is a specific EP2-receptor agonist. We have shown that butaprost has very low inhibitory activity on the guinea-pig vas deferens, a very sensitive EP3-receptor containing preparation. However, on the chick ileum, the original EP3 preparation, butaprost showed potent contractile activity (pEC25 approximately 8.0).In addition, its maximum response was lower than that of PGE2; lower maxima were also found for sulprostone, MB 28767 and oxoprostol, but not for ICI 80205, 16,16-dimethyl PGE2 and 17-phenyl-omega-trinor PGE2. The maximal response to a combination of either sulprostone and butaprost or sulprostone and PGE2 was similar to that achieved by PGE2 alone. Analysis of the interaction between sulprostone and PGE2 appears to exclude a partial agonist action for sulprostone. Furthermore neither sulprostone nor butaprost appear to have inhibitory activity on the ileum. AH 6809 at 2 pM produced only a small shift of the PGE2 log concentration-response curve.5. It is likely that contraction of the longitudinal smooth muscle of the chick ileum is mediated by (at least) two EP-receptor subtypes; activation of only one receptor system does not induce the maximum response (i.e. the acetylcholine maximum) of the preparation. One receptor could be an EP3 subtype, at which sulprostone exerts a selective agonist action. The other receptor is unlikely to be an EP, subtype, because of the high agonist potency of butaprost, the low agonist potency of iloprost, and the low antagonist potency of AH 6809. An alternative hypothesis is that the chick ileum contains a novel EP-receptor subtype in addition to an EP3-receptor.