BACKGROUND AND PURPOSE: A single dose of JV-GL1 lowers intraocular pressure (IOP) for a week according to previous studies on non-human primates. This highly protracted effect did not correlate with its ocular bio-disposition, where the drug was undetectable inside the eye after only one day post-dosing. Our current studies were intended to determine the role of EP2 receptors in mediating the long-term ocular hypotensive activity of JV-GL1 and utilized mice deficient in EP2 receptors. EXPERIMENTAL APPROACH: The protracted intraocular pressure reduction produced by JV-GL1 was investigated in C57BL/6J and EP2 receptor knock-out mice (B6.129-Ptger2tm1Brey/J, EP2KO). Both ocular normotensive and steroid induced ocular hypertensive (SI-OHT) mice were studied. Intraocular pressure was measured tonometrically under general anesthesia. Aqueous humor outflow facility was measured ex vivo using the iPerfusion system 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. KEY RESULTS: In SI-OHT, JV-GL1 did not lower IOP in EP2 KO mice. However, in WT mice with SI-OHT, JV-GL1 lowered IOP for 4-6 days. JV-GL1 did not alter outflow facility in WT mice at 3 days after topical administration. CONCLUSIONS AND IMPLICATIONS: The long-term effect of JV-GL1 on IOP in the SI-OHT model of glaucoma is EP2 receptor dependent. Such protracted activity of a single dose of a small molecule (JV-GL1) is unprecedented. Future studies on JV-GL1 may eventually lead to “once-weekly” small molecules, with reduced drug prices and better disease control.