Mature glomerular visceral epithelial cells, or podocytes, are unique cells with a complex cell architecture. Characteristically, they possess a highly branched array of major processes and foot processes, which are essential for glomerular filtration in the kidney. A podocyte cell line with the potential to exhibit many features of differentiated podocytes, particularly the formation of cell processes, was recently established. In this study, it is shown that directed membrane transport is involved in process formation in cultured podocytes. The well-characterized vesicular stomatitis virus G was used as a marker protein for the biosynthetic pathway in these cells. It seems that newly synthesized vesicular stomatitis virus G is preferentially delivered into the cell processes of the podocytes, where it is colocalized with known regulators of vesicular transport from the Golgi apparatus to the plasma membrane, such as the small GTPase rab8 and the sec6/sec8 complex. To determine the role of vesicular transport in process formation, cells were treated with brefeldin A, a drug that disrupts the trafficking of post-Golgi transport vesicles. As a result, the podocytes reversibly lost their ability to form processes. These findings suggest that podocytes are dependent on a constant fresh source of lipids and proteins to form their processes.