We previously reported that the percentage of reversibly and irreversibly sickled cells (RSC and ISC, respectively) in the blood of patients with sickle cell disease is strongly influenced by the method of blood drawing (PNAS 91:12589, 1994). We now document the effect of blood storage conditions on the percentage of RSC and ISC. The percentage of RSC was lowest when blood was stored at 0 degree C, while the percentage of RSC was highest in specimens kept at 37 degrees C. At room temperature, the percentage of RSC increased slightly over 8 hours. The percentage of ISC was also temperature dependent and was reduced significantly upon cooling. Our results showed that many ISC reverted to a discoidal shape after 3 hrs of cooling after treatment of blood with oxygen or carbon monoxide. Since no Hb S polymers were detected in ISC treated with oxygen or carbon monoxide, the time required for shape restoration may be attributed to the membrane. We measured ISC levels of 10 patients with consideration of storage temperature and compared the values with those determined by the conventional method and also with those published previously.