The reduction of urinary volume after the use of thiazide in the treatment of diabetes insipidus (DI) is known as the "paradoxical effect." Since enhanced proximal solute and water reabsorption only partially account for the reduction in urinary volume, an additional diuretic effect on nephron terminal segments was postulated. Thus the aim of our work was to investigate the effect of hydrochlorothiazide (HCTZ) on water transport in the inner medullary collecting duct (IMCD) of normal and Brattleboro rats. Osmotic water permeability (P_f) and diffusional water permeability (P_dw) were studied at 37°C and pH 7.4 by the in vitro microperfusion technique. In the absence of antidiuretic hormone (ADH), HCTZ (10⁶ M) added to the perfused fluid enhanced P_f from 6.36 ± 0.56 to 19.08 ± 1.70 µm/s (P < 0.01) and P_dw from 38.01 ± 4.52 to 52.26 ± 4.38 ×10⁵ cm/s (P < 0.01) in normal rats and also stimulated P_f in Brattleboro rats from 3.53 ± 1.41 to 11.16 ± 1.13 µm/s (P < 0.01). Prostaglandin E₂ (PGE₂) (10⁵ M) added to the bath fluid inhibited HCTZ-stimulated P_f (in µm/s) as follows: control, 16.93 ± 2.64; HCTZ, 29.65 ± 5.67; HCTZ+PGE₂, 10.46 ± 1.84 (P < 0.01); recovery, 16.77 ± 4.07. These data indicate that thiazides enhance water absorption in IMCD from normal rats (in the absence of ADH) and from Brattleboro rats and that the HCTZ-stimulated P_f was partially blocked by PGE₂. Thus we may conclude that the effect of thiazide in the treatment of DI occurs not only in the Na⁺-Cl cotransport in the distal tubule but also in the IMCD.