Background: The mechanism of renal stone genesis as well as the location of stone crystal formation in the kidney remains unclear. Possible sites of stone generation are either in the tubular lumen or tubular cell.Methods: We cultured Madin Darby canine kidney (MDCK), LLC‐PK1 and Magen Krebs Niigata‐28 (MKN‐28) cells in DMEM + 10% FBS medium in a well without passage for 30 days.Results: MDCK cells produced microliths at the basolateral side but not on the lumen side of these cells. The other two cell lines did not form microliths.Conclusion: Our data show that microlith formation is a characteristic of MDCK cells and that biological mineralization of MDCK cells may serve as a human urolithiasis model in vitro. The findings support a significant role of the renal distal convoluted tubule and collecting ducts in the in vitro generation of urinary s