Storage time for platelet concentrates (PCs) is limited to five days due to 'aging' of the platelets and an increasing risk of bacterial proliferation. Storage time can be prolonged by cryopreservation. We investigated in vitro function of six consecutive PCs at the end of their conventional shelf life followed by cryopreservation for 24 hours. Spontaneous, adenosine diphosphate (ADP)-induced and collagen-induced activation before and after cryopreservation were determined by flow cytometry. Additionally, ADP- and collagen-induced aggregation was measured. After cryopreservation two-thirds of the platelets were spontaneously activated, twice as many as before the procedure (p < 0.001). ADP-induced activation was significantly reduced (p = 0.014). Collagen-induced activation was unchanged. Aggregation stimulated by ADP and collagen was significantly reduced (p = 0.005 and p = 0.009, respectively). Our results show severely impaired in vitro function of platelets after storage at 22 degrees C for five days followed by cryopreservation. Cryopreservation of PCs after a storage time of five days cannot be recommended.