The tubular effects of prostaglandin (PG) E2 on electrolyte and water excretion were investigated in vitro in the nonanesthetized chicken by the Sperber technique. This technique allowed the administration of PGE2 directly into the peritubular space of one kidney by way of the venous portal circulation. When compared to the contralateral, noninfused kidney, PGE2 in the infused kidney (0.6-4.5 X 10(-10) mol/kg X min) induced a dose-dependent increase in urinary flow rate, a mild natriuresis and kaliuresis, with a concomitant decrease in urinary osmolality and an increase in free-water clearance. These effects occurred without changes in renal plasma flow or glomerular filtration rate. PGA2 (1.7-7.8 X 10(-10) mol/kg X min), another vasodepressor PG, did not modify electrolyte excretion. The tubular handling of PGE2 was observed by following the administration of [3H]PGE2. [3H] PGE2 was metabolized extensively during its renal tubular excretion. The 3H label was secreted actively into the urine by the organic anion transport system which was inhibited by novobiocin. Inhibition of the organic anion transport system did not modify the renal tubular effects of PGE2 on electrolyte and water excretion. These results indicate that PGE2 exerts a tubular inhibitory effect on sodium and water excretion, this action being located on the peritubular side.