Mature podocytes are among the most complex differentiated cells and possess a highly branched array of foot processes that are essential to glomerular filtration in the kidney. Such differentiated podocytes are unable to replicate and culturing of primary podocytes results in rapid growth arrest. Therefore, conditionally immortalized mouse podocyte clones (MPC) were established, which are highly proliferative when cultured under permissive conditions. Nonpermissive conditions render the majority of MPC cells growth arrested within 6 days and induce many characteristics of differentiated podocytes. Both proliferating and differentiating MPC cells express the WT-1 protein and an ordered array of actin fibers and microtubules extends into the forming cellular processes during differentiation, reminiscent of podocyte processes in vivo. These cytoskeletal rearrangements and process formation are accompanied by the onset of synaptopodin synthesis, an actin-associated protein marking specifically differentiated podocytes. In addition, focal contacts are rearranged into an ordered pattern in differentiating MPC cells. Most importantly, electrophysiological studies demonstrate that differentiated MPC cells respond to the vasoactive peptide bradykinin by changes in intracellular calcium concentration. These results suggest a regulatory role of podocytes in glomerular filtration. Taken together, these studies establish that conditionally immortalized MPC cells retain a differentiation potential similar to podocytes in vivo. Therefore, the determinative steps of podocyte differentiation and process formation are studied for the first time using an inducible in vitro model.