We have carried out LC-MS/MS-based proteomic profiling of differential centrifugation fractions from rat inner medullary collecting duct (IMCD): 1) to provide baseline knowledge of the IMCD proteome; and 2) to evaluate the utility of differential centrifugation in assessing trafficking of the water channel aquaporin-2 (AQP2). IMCD suspensions were freshly prepared from rat kidneys using standard methods. Homogenized samples were subjected to sequential centrifugations at 1,000 g, 4,000 g, 17,000 g and 200,000 g. These samples, as well as the final supernatant, were subjected to LC-MS/MS analysis. Preliminary immunoblotting confirmed that the ratio of AQP2 in the 17,000 g fraction to the 200,000 g fraction underwent an increase in response to the vasopressin analog dDAVP, largely due to a reduction in the 200,000 g fraction. Immunoblotting for the major phosphorylated forms of AQP2 revealed that phosphorylated AQP2 was present in both the 17,000 g and 200,000 g fractions. LC-MS/MS analysis showed that markers of 'intracellular vesicles', chiefly endosomal markers, were present in both the 17,000 g and the 200,000 g fractions. In contrast, plasma membrane proteins were predominantly present in the 4,000 g and 17,000 g fractions. Proteins associated with several multiprotein complexes (e.g. actin-related protein 2/3 complex and proteasome complex) were virtually exclusively present in the 200,000 g fraction. Overall, we identified 656 proteins, including 189 not previously present in the IMCD database. The data show that both the 17,000 g and 200,000 g fractions are highly heterogeneous and cannot be equated with 'plasma-membrane' and 'intracellular vesicle' fractions, respectively, leading us to propose an alternative approach for use of differential centrifugation to assess vesicular trafficking to the plasma membrane.