One of the obstacles in analyzing frozen urine samples is the formation of uncharacterized precipitates. Frequently, these precipitates are discarded prior to analysis. Some laboratory data may be erroneous if these precipitates contain important compounds. In the present study, we examined urinary sediments precipitated after an overnight storage at -20°C. Although cells and debris were removed prior to freezing, the precipitates remained, whereas storing the centrifuged urine overnight at 4°C did not result in precipitate formation. There were no significant differences observed among 10 healthy individuals (5 males and 5 females). EDTA (5 mM) could efficiently reduce the amount of precipitates to approximately 25% of the initial amount. The addition of exogenous CaCl₂, but not NaOx and NaCl, significantly increased the amount of precipitates in a dose-dependent manner. Linear regression analysis revealed a significant correlation between endogenous urinary calcium level and the amount of precipitates (r=0.894; p<0.001). Urine pH also had some effects on type and amount of precipitates. These precipitates were composed mainly of calcium oxalate dihydrate and amorphous calcium crystals. The results also showed that these precipitates could deplete urinary proteins and calcium ions (23.6±1.1 % decrease). Therefore, these freezer-induced urinary sediments significantly affect protein analysis and measurement of calcium levels in the urine. However, vigorous shaking of the sample at room temperature could re-dissolve these precipitates. Our data strongly indicate that these freezer-induced precipitates must be taken into account when the frozen urine samples are analyzed.