One of the recent approaches to enhance desiccation tolerance in red blood cells (RBCs) is by loading trehalose. This process has been shown to increase the recovery of lyophilized RBCs; conversely, it results in cellular damage including hemoglobin oxidation and loss of membrane integrity. The purpose of this study was to further investigate the extent of oxidative injury during the loading of trehalose into RBCs. RBCs were incubated in the absence (control) or presence of trehalose (0.8 mol/l) at 4 degrees C or 37 degrees C for different time scales. Oxidative damage was monitored by flow cytometry using dichlorofluorescin for reactive oxygen species formation, Annexin V-FITC for phosphatidylserine translocation and fluorescein-DHPE for lipid peroxidation. Percent methemoglobin, percent hemolysis and thiobarbituric acid reactive substances were measured by spectrophotometry. The extent of oxidative damage during trehalose loading is affected by the incubation temperature, incubation time and the presence of trehalose. Incubation at 4 degrees C was relatively innocuous; however, oxidative injury was evident at 37 degrees C in both RBC groups. The addition of trehalose is correlated with high osmotic pressure, which had minor effects during incubation at 4 degrees C, but seemed to have exacerbated the severity of cellular injury at 37 degrees C, as measured by higher levels of hemolysis, methemoglobin and lipid peroxidation. The process of trehalose-loading is problematic due to its requirement for prolonged incubations at 37 degrees C. These conditions are correlated with oxidative injury, even in the absence of trehalose. While trehalose is believed to be crucial for stabilizing biomembranes, the consequences of its introduction into the cells require further investigation.