Discrimination of EP₁ and EP₃ prostanoid receptors has mainly relied on the use of selective agonists in combination with EP₁ antagonists, although EP₃ antagonists are now emerging. We were concerned about the lack of functional data for two newer agonists, ONO-DI-004 (EP₁-selective) and ONO-AE-248 (EP₃-selective) (Suzawa et al., 2000). Consequently, we have examined these agents on guinea-pig trachea (EP₁ preparation) and guinea-pig aorta and rat mesenteric artery (EP₃ preparations). Ring preparations obtained from male guinea-pigs (600 – 800 g) and male rats (250 – 300 g) were suspended in Krebs-Henseleit solution (95% O₂ / 5% CO₂) at 37^oC in 10 ml tissue baths. Tension was recorded with a force transducer and relayed to AD Instruments Chart software. Cumulative agonist sequences were obtained in the presence of the COX inhibitor indomethacin (1 µM) and the TP antagonist BMS-180291 (300 nM), and under priming with 20 mM K⁺ on guinea-pig aorta. The agonist potencies of 17-phenyl PGE₂ (EP₁ > EP₃), sulprostone (EP₃ > EP₁), ONO-DI-004 and ONO-AE-248 are shown in Table 1. EC₅₀ values for 17-phenyl PGE₂ on trachea were 0.74 – 1.3 – 2.0 nM (mean / range) and for sulprostone on aorta and mesenteric artery were 0.079 – 0.29 – 0.87 nM and 7 – 11 – 16 nM respectively. ONO-AE-248 had a shallower log concentration-response curve than sulprostone on guinea-pig aorta. The EP₁ antagonists SC-51322 (0.03 – 1 µM) and AH-6809 (0.1 – 3 µM) abolished established responses to 10 nM 17-phenyl PGE₂ / 1.4 µM ONO-DI-004 on guinea-pig trachea; pA₂ values computed by the Gaddum method were 8.09 ± 0.05 / 7.99 ± 0.10 and 7.61 ± 0.04 / 7.48 ± 0.02 (± SEM, n = 4) respectively. Comparable studies are in progress with an EP₃ antagonist. It is clear that ONO-DI-004 and ONO-AE-248 show high selectivity for EP₁ and EP₃ receptors respectively. However, this occurs at the expense of considerable loss of potency, to the extent that it would be difficult to use these agonists in a Schild antagonism protocol. They could be used in a Cheng-Prusoff protocol (Leff & Dougall, 1993), with the advantage that their onset and offset of action are faster than those of 17-phenyl PGE₂ and sulprostone, which probably reflects their lower potencies / affinities.