Background In pretransfusion testing at our institution, unexplained reactions are reported as antibody of undetermined specificity (AUS) after antibodies against Food and Drug Administration-specified red blood cell antigens have been ruled out. The frequency, laboratory features, and natural history of these reactions are not well defined. Study Design and Methods We retrospectively examined AUS reported at a single institution between July 1, 2009, and December 31, 2011. For AUS reported in the first quarter of 2012, the reference workup and antibodies identified during subsequent testing were reviewed to characterize the laboratory features and natural history of AUS. Results A total of 8121 antibodies were detected in 6058 patients during the study period. AUS was reported 1442 times (18%) and was the single most reported event, followed by anti-E (18%) and anti-K (14%). In the first quarter of 2012, AUS was reported in 174 unique patients. Most AUS (78%) reacted with two cells or less tested in gel and most reactions (98%) were 1+ or weaker. Forty-five patients presenting with AUS for the first time had repeated antibody workup later. AUS persisted in 31 cases for 2 to 60 days. AUS disappeared in 14 cases, seven of which developed a total of 10 new antibodies (three anti-E; one anti-D; one anti-C; two anti-Jkb; and one each of anti-Lea, anti-s, and warm autoantibody) in 3 to 21 days (median, 8 days). Conclusion AUS is a common finding with our pretransfusion testing method. These reactions are heterogeneous and may represent antibodies against low-prevalence antigens, non-RBC antigens, or developing antibodies that are clinically significant.