BACKGROUND The relationship between length of storage of red blood cell (RBC) units and biochemical changes has been well studied, but little is known about the progression of cellular immunomodulative properties in blood recipients. This study aims to quantify in vitro T-cell activation and cytokine release by white blood cells, after incubation with supernatants from leukoreduced RBCs. STUDY DESIGN AND METHODS Whole blood cultures were incubated with supernatant from five leukoreduced RBC units stored for 1, 6, 10, 15, 24, and 42 days. Supernatant-induced T-cell activation was evaluated by quantifying CD25 expression. Supernatant-induced cytokine production was determined by measuring interleukin (IL)-6, IL-10, and tumor necrosis factor (TNF)-alpha levels. RESULTS No cytokines were detected in RBC supernatants even after 42 days of storage. However, IL-6 levels in whole blood culture increased significantly when incubated with supernatant from RBC units stored for 1, 6, and 15 days, by factors of 1.7 +/- 0.3, 1.7 +/- 0.3, and 1.4 +/- 0.3, respectively. TNF-alpha levels were significantly decreased on Days 24 and 42 of storage by factors of 0.50 +/- 0.42 and 0.33 +/- 0.21, respectively. IL-10 levels were significantly increased on Days 1 and 42 of storage by factors of 2.3 +/- 1.3 and 3.2 +/- 2.8, respectively. After an initial increase in IL-6 and TNF-alpha production, there was a significant linear decrease in their levels measured from units stored for longer times. No significant changes in CD25 expression were observed over time. CONCLUSION Although no cytokines were measured in the supernatants from leukoreduced RBCs, these supernatants exhibited variable immunomodulatory effects related to their length of storage.