Background: Intravital microscopy theoretically provides the optimal conditions for studying specific organ functions. However, the application of microscopy in intact organs in vivo has been limited so far due to technical difficulties. The purpose of this study was to establish a method of in vivo confocal laser scanning microscopy (CLSM) for the study of endocytosis in proximal tubules of intact kidney. Methods: The left kidney of rats placed on a modified microscope stage was exposed and stabilized in a thermostatically controlled cup. The stage was then attached to an upright confocal microscope. Surface proximal tubules were microinfused with fluorescent albumin or transferrin. Single or time-series images of microinfused proximal tubules were recorded in reflection and/or fluorescence mode. Results: The stability of the kidney and the resolution of images were sufficient to visualize intracellular vesicles. Albumin and transferrin were initially observed at the brush border, then later internalized by proximal tubules and accumulated in lysosomes over a time period of 15 min. Furthermore, fusion of vesicles was observed in time-lapse images. Conclusion: These results show that in vivo CLSM in intact kidney may be an excellent method to evaluate proximal tubular endocytosis and ligand trafficking.