BACKGROUND Calcium levofolinate infusions could be prepared in advance by a centralized intravenous additive service (CIVAS) to improve safety and time management. OBJECTIVE To investigate the effect of freezing, microwave thawing and long-term storage at 5 +/- 3 degrees C on the stability of calcium levofolinate in 5% dextrose solution. METHODS Solutions of 250 mL of 5% dextrose in polyolefin bags (n = 5) containing approximately 400 mg of calcium levofolinate were prepared under aseptic conditions and frozen for 95 days at -20 degrees C. The solutions were then thawed using microwaves and stored at 5 +/- 3 degrees C for 1 month. The calcium levofolinate concentrations were measured by high performance liquid chromatography (HPLC). Visual inspection was performed and pH was measured periodically during the storage at 5 +/- 3 degrees C. Stability of the solution was defined as a concentration remaining superior to 90% of the initial concentration by regression analysis as recommended by the Food and Drug Administration (FDA). RESULTS No colour change or precipitation in the solutions was observed. Calcium levofolinate infusions were stable when stored at 5 +/- 3 degrees C during 1 month after freeze-thaw treatment. Throughout this period, the lower confidence limit of the estimated regression line of concentration-time profile remained above 90% of the initial concentration. Slight change in pH values from 6.52 +/- 0.01 to 6.50 +/- 0.01 during storage time did not affect retention time on HPLC and has no clinical consequence, the solutions remaining in the acceptable range for perfusion (4 <or= pH <or= 10). CONCLUSION Under the conditions of this study, calcium levofolinate in 5% dextrose infusion may be prepared, frozen in advance by CIVAS, and then microwave thawed before use. Such treatment extends long-term stability and releases pharmacist's time for major activities such as checking medication order errors.