Cold agglutinins (CA) were evaluated prospectively in patients with various mononucleosis syndromes and in a large control group. Cold agglutinins with anti-i specificity were seen mainly in heterophil- positive or -negative Epstein-Barr virus (EBV)-induced infectious mononucleosis (31.8% of cases). Unclassified CA with equal reactivity against cord and adult erythrocytes were seen in 56 of 150 (37.3%) cases of heterophil-antibody-positive infectious mononucleosis (IM), in 1 of 7 (14.3%) cases of heterophil-negative EBV-induced IM, and in 12 of 31 (38.7%) cases of the heterophil-negative mononucleosis-like syndrome due to cytomegalovirus or other unspecified agents. One patient with heterophil-positive IM had a persistent, partially papain sensitive CA with anti-Pr-like activity. Anti-i CA were seen in less than 1.0% of healthy young adults (500) or patients without mononucleosis (500) submitted for heterophil studies. Unclassified CA were noted in 3.2% of the latter 1000 samples.