Transfusion-associated graft-versus-host disease (TA-GVHD) is usually a fatal outcome of blood transfusion therapy, caused by viable leucocytes contained in the donor blood. Most cases of TA-GVHD occur when less than 4-d-old blood is transfused. We therefore examined the molecular changes that occur during storage that may account for the paucity of TA-GVHD following infusion of older blood. Leucocyte number and viability were essentially unchanged from freshly obtained blood, but the expression of cell-surface lymphocyte activation antigens (CD3, CD4, CD28, CD2, CD45) decreased rapidly within the first 24 h and continued to fall to less than 20% of original levels by d 9 of storage at 4 degrees C. The decrease in CD antigen expression directly correlated with a decreasing ability to induce activation of the T-lymphocyte cellular signal transduction pathway. As a result, cells became less responsive in a mixed lymphocyte culture (MLC) by d 3, with abrogation of the MLC responsiveness by d 5. Donor leucocytes stored for 4 d or less at 4 degrees C were able to partially re-express CD antigens and reconstitute their signalling pathway when placed at 37 degrees C. whereas those stored for more than 4 d were not. These irreversible changes result from a permanent downregulation of donor cell protein synthesis. These findings provide a mechanism to explain the paucity of TA-GVHD following transfusion of blood that is more than 4 d-old. Further study may show that aged blood provides additional assurances for the prevention of TA-GVHD; however, use of aged blood should not replace current protocols using irradiation.