Long-term storage of platelets is infeasible due to platelet activation at low temperatures. In an effort to address this problem, we evaluated the effectiveness of a formulation combining trehalose and phosphate in protecting platelet structure and function following cryopreservation. An annexin V binding assay was used to quantify the efficacy of the trehalose and phosphate formulation in suppressing platelet activation during cryopreservation. Of the platelets cryopreserved with the trehalose plus phosphate formulation, 23% ± 1.2% were nonactivated, compared with 9.8% ± 0.26% nonactivated following cryopreservation with only trehalose. The presence of both trehalose and phosphate in the cryopreservation medium is critical for cell survival and preincubation in trehalose plus phosphate solutions further enhances viability. The effectiveness of trehalose plus phosphate in preserving platelets in a nonactivated state is comparable to 6% dimethyl sulfoxide (Me2SO). Measurements of platelet metabolic activity using an alamarBlue assay also established that trehalose plus phosphate is superior to trehalose alone. Finally, platelets protected by the trehalose plus phosphate formulation exhibit similar aggregation response upon thrombin addition as fresh platelets, but an increase of cytosolic calcium concentration upon thrombin addition was not observed in the cryopreserved platelets. These results suggest that trehalose and phosphate protect several aspects of platelet structure and function during cryopreservation, including an intact plasma membrane, metabolic activity, and aggregation in response to thrombin, but not intracellular calcium release in response to thrombin. © 2005 Wiley Periodicals, Inc.