Occasional units of red blood cells develop a positive direct antiglobulin test (DAT) when frozen and deglycerolyzed by the Meryman method. The presence of cold autoagglutinins with a high titer at 4 degrees C or autoreactivity at room temperature were generally predictive of which units would develop a positive DAT, but neither was reliable in all cases. An indicator system is presented in which a DAT is performed on a segment that has been glycerolized and deglycerolized in a manner similar to the Meryman method for freezing a donor unit. This system is extremely reliable in predicting which units will convert from DAT-negative to DAT-positive during processing. The positive DAT of deglycerolized red blood cells was due to C4 and weak C3d binding. This complement binding can be prevented by addition of ethylenediaminetetraacetic acid (EDTA) or removal of plasma before the addition of glycerol.