The use of the direct manual hexadimethrine bromide (Polybrene) test (DPT) in the investigation of patients for autoimmune hemolytic anemia (AIHA) was evaluated. Seventy-nine blood samples from 68 patients were tested. A direct antiglobulin test (DAT) using monospecific reagents and the DPT were performed, and a concentrated ether eluate was tested. The DAT was positive in 62 (78%) of 79 patients and negative in 17 (22%). There is a good correlation among DAT, eluate, and DPT in demonstrating the presence of immunoglobulin on the red cell surface. In contrast, the DPT does not detect C3d and is often negative in cases of AIHA in which C3d alone is demonstrated by the DAT. In DAT-negative cases, DPT results correlated with reactive eluates. However, in four cases of steroid-responsive, DAT-negative hemolytic anemia, the DPT supported the diagnosis of AIHA when the eluate did not react. The DPT is a useful additional screening test for the investigation of AIHA, but it is not recommended as a replacement for either eluate testing or the DAT.