Podocytes, cells of the visceral epithelium in the kidneys, are a key component of the glomerular filtration barrier. Podocyte damage and loss contribute to the initiation of glomerular diseases. NIH investigators recently established long-term urinary cell cultures from two patients with focal segmental glomerulosclerosis and two healthy volunteers, via transformation with the thermosensitive SV40 large T antigen (U19tsA58) together with human telomerase (hTERT). Characterization of randomly selected clonal cell lines from each human subject showed mRNA expression for the podocyte markers synaptopodin, nestin, and CD2AP in all clones. Podocin mRNA was absent from all clones. The expression of nephrin, Wilms tumor 1 (WT1), and podocalyxin mRNA varied among the clones, which may be due to transformation and/or cloning. These novel human urine-derived podocyte-like epithelial cell lines (HUPECs) generated from urine of patients and healthy volunteers will be useful to study podocyte cell biology.