PGE₂ has an established role in renal water handling. The present study was undertaken to examine the role of mcrosomal prostaglandin E synthase-1 (mPGES-1) in the diuretic response to acute and chronic water loading. As compared with wild type (+/+) controls, mPGES-1 -/- mice exhibited impaired ability to excrete an acute, but not chronic water load. In response to acute water loading, urinary PGE₂ excretion in the +/+ mice increased at 2 h, in parallel with increased urine flow. In contrast, the -/- mice exhibited a delayed increase in urinary PGE2 excretion, coinciding with the stimulation of renal medullary mRNA expression of cytosolic prostaglandin E synthase (cPGES) but not mPGES-2. At baseline, renal aquaporin-2 (AQP2) expression in mPGES-1 -/- mice was enhanced as compared to the +/+ control. In response to acute water loading, renal AQP2 expression in the +/+ mice was significantly reduced and this reduction was blunted in the -/- mice. Despite striking changes in AQP2 protein expression, renal AQP2 mRNA in both genotypes largely remained unchanged. Overall, these data support an important role of mPGES-1 in provoking the diuretic response to acute water loading.