A single application of JV-GL1 substantially lowers non-human primate intraocular pressure (IOP) for about a week, independent of dose. This highly protracted effect does not correlate with its ocular biodisposition or correlate with the once-daily dosing regimen for other prostanoid EP receptor agonists such as trapenepag or omidenepag. The underlying pharmacological mechanism for the multiday extended activity of JV-GL1 is highly intriguing. The present studies were intended to determine EP receptor involvement in mediating the long-term ocular hypotensive activity of JV-GL1 by using mice genetically deficient in EP receptors. The protracted IOP reduction produced by JV-GL1 was investigated in C57BL/6J and EP receptor knock-out mice (B6.129- /J; EP KO). Both ocular normotensive and steroid-induced ocular hypertensive (SI-OHT) mice were studied. IOP was measured tonometrically under general anaesthesia. Aqueous humour outflow facility was measured ex vivo using in normotensive C57BL/6J mouse eyes perfused with 100 nM de-esterified JV-GL1 and in SI-OHT C57BL/6J mouse eyes that had received topical JV-GL1 (0.01%) 3 days prior. Both the initial 1-day and the protracted multiday effects of JV-GL1 in the SI-OHT model for glaucoma were abolished by deletion of the gene encoding the EP receptor. Thus, JV-GL1 did not lower IOP in SI-OHT EP KO mice, but in littermate SI-OHT EP WT control mice, JV-GL1 statistically significantly lowered IOP for 4-6 days. Both the 1-day and the long-term effects of JV-GL1 on IOP are entirely EP receptor dependent.