I t has been controversial whether high water permeability in the thin descending limb of Henle (TDLH) is required for formation of a concentrated urine by the kidney. Freeze-fracture electron microscopy (FFEM) of rat TDLH has shown an exceptionally high density of intramembrane particles (IMPs), which were proposed to consist of tetramers of aquaporin-1 (AQP1) water channels. In this study, transepithelial osmotic water permeability (P_f) was measured in isolated perfused segments (0.5–1 mm) of TDLH in wild-type (+/+), AQP1 heterozygous (+/–), and AQP1 null (–/–) mice. P_f was measured at 37°C using a 100 mM bath-to-lumen osmotic gradient of raffinose, and fluorescein isothiocyanate (FITC)–dextran as the luminal volume marker. P_f was (in cm/s): 0.26 ± 0.02 ([+/+]; SE, n = 9 tubules), 0.21 ± 0.01 ([+/–]; n = 12), and 0.031 ± 0.007 ([–/–]; n = 6) (P < 0.02, [+/+] vs. [+/–]; P < 0.0001, [+/+] vs. [–/–]). FFEM of kidney medulla showed remarkably fewer IMPs in TDLH from (–/–) vs. (+/+) and (+/–) mice. IMP densities were (in μm^–2, SD, 5–12 micrographs): 5,880 ± 238 (+/+); 5,780 ± 450 (+/–); and 877 ± 420 (–/–). IMP size distribution analysis revealed mean IMP diameters of 8.4 nm ([+/+] and [+/–]) and 5.2 nm ([–/–]). These results demonstrate that AQP1 is the principal water channel in TDLH and support the view that osmotic equilibration along TDLH by water transport plays a key role in the renal countercurrent concentrating mechanism. The similar P_f and AQP1 expression in TDLH of (+/+) and (+/–) mice was an unexpected finding that probably accounts for the unimpaired urinary concentrating ability in (+/–) mice.