Objectives To date, there is only one study investigating the physicochemical stability of diluted mitomycin (MMC) solutions prepared by using urea-containing Mitomycin medac as starting material. The aim of the study was to test the solubility of the new MMC formulation with regard to highly concentrated solutions and determine the physicochemical stability of clinically relevant MMC concentrations stored under different conditions in various primary containers. Methods Mitomycin medac was dissolved with water for injection to achieve MMC concentrations of 1 mg/mL and 2 mg/mL. Additionally, 1 mg/mL MMC solutions were further diluted with normal saline to obtain 0.2 mg/mL and 0.4 mg/mL solutions. According to clinical practice, 1 mg/mL solutions were stored in original glass vials and 2 mg/mL, 0.2 mg/mL, and 0.4 mg/mL solutions were stored in plastic syringes. All solutions were stored either refrigerated or at 20–25 °C light protected for up to 8 days. Samples were taken immediately after dissolution or dilution and at predetermined time points. Physicochemical stability was determined by reversed-phase high-performance liquid chromatography (RP-HPLC) with photodiode array detection, pH and osmolality measurement, and inspection for visible particles or color changes. Results 2 mg/mL MMC solutions were achieved at room temperature and physicochemical stability was given for 8 h, independent of the storage temperature. Between 8 and 12 h of storage, crystallization occurred in almost all samples. In 1 mg/mL MMC test solutions, stored under refrigeration, crystallization occurred in 2 of 3 vials after 2 and 4 days of storage, respectively. In the vial without signs of crystallization, MMC concentration amounted to >90 % of the initial measured concentration after 6 days. When stored at room temperature, crystallization was not seen, but MMC concentration declined below the 90 % stability limit at about 15 h of storage. In 0.2 mg/mL and 0.4 mg/mL MMC test solutions crystallization was not observed at all. When stored refrigerated, preparations were physicochemically stable for 5 and 3 days, respectively. When stored light protected at room temperature, physicochemical stability was given for at least 6 h, irrespective of the MMC concentration. Conclusions Mitomycin medac enables the preparation of 2 mg/mL MMC solutions without additional heating and shaking due to the hydrotropic activity of urea contained as excipient. However, in 2 mg/mL MMC solutions crystallization is the most dominant stability limiting factor, especially under refrigerated storage. Hence, storage at room temperature is recommended for this concentration. In 1 mg/mL MMC solutions crystallization is less prominent. To avoid increased chemical degradation at room temperature, refrigerated storage is recommended. Both, 2 mg/mL and 1 mg/mL MMC solutions should always be checked for the formation of crystals before use. In diluted 0.2 mg/mL and 0.4 mg/mL MMC solutions, crystallization was not an issue and refrigerated storage extends physicochemical stability to maximum 5 and 3 days, respectively.