The increasing number of available genetically manipulated mice makes it necessary to develop tools and techniques for examining the phenotypes of these animals. We have developed a straightforward and rapid method for the isolation of large quantities of single tubule fragments from the mouse kidney. Immunohistochemistry, electron microscopy, and fluorescence microscopy were used to evaluate the viability, functional characteristics, and morphology of proximal tubules (PT), and collecting ducts from cortex (CCD) and inner stripe of the outer medulla (ISOMCD). Tubules were isolated using a modified collagenase digestion technique, and selected under light microscopy for experimentation. Electron microscopy and trypan blue exclusion showed that a large portion of unselected proximal tubules were damaged by the digestion procedure. The selected tubules, however, all excluded trypan blue, indicating that the plasma membrane had remained intact. Immunocytochemistry on isolated CCD showed normal distribution of H⁺-ATPase, pendrin, and anion exchanger-1 (AE-1) staining. The pH-sensitive dye 2′,7′-bis(2-carboxylethyl)-5(6)-carboxyfluorescein (BCECF) was used to measure Na⁺-dependent and -independent intracellular pH (pH_i) recovery rates in PT, and in single intercalated cells of CCD and ISOMCD fragments. Na⁺-dependent pH_i-recovery was 0.144±0.008 (PT), 0.182±0.013 (CCD), and 0.112±0.010 pH units/min. (ISOMCD). Na⁺-independent pH_i recovery was found in all three segments (PT: 0.021±0.002, CCD: 0.037±0.002, ISOMCD: 0.033±0.002 pH units/min) and was sensitive to concanamycin. In summary, we have developed a new technique for rapid and straightforward preparation of large quantities of defined tubule fragments from mouse kidney. Using this technique, the first measurements of plasma membrane vacuolar H⁺-ATPase activities in mouse PT and collecting duct were made. This technique will facilitate further characterization of kidney function in normal and genetically manipulated animals.