BACKGROUND First-generation cephalosporins rarely caused immune hemolytic anemia (IHA). Second- and third-generation cephalosporins, especially cefotetan and ceftriaxone, are increasingly associated with severe, sometimes fatal IHA. STUDY DESIGN AND METHODS Samples from 53 patients with drug-induced IHA and/or positive direct antiglobulin test (DAT) were tested. Patients' sera were tested against drug-treated red cells (RBCs) and untreated or enzyme-treated RBCs, with and without the addition of drug solution. Eluates from patients' RBCs were tested against drug-treated and untreated RBCs. RESULTS Forty-three patients had antibodies to cefotetan, 8 to ceftriaxone, 1 to cefoxitin, and 1 to cefotaxime. All patients had a positive DAT; only anticefoxitin and anti-cefotetan were demonstrable in RBC eluates. Sera containing anti-cefoxitin, anti-cefotaxime, and anti-cefotetan reacted with drug-treated RBCs (100%) and untreated or enzyme-treated RBCs in the presence of drug (98% or 100%, respectively). All of the ceftriaxone antibodies reacted with untreated or enzyme-treated RBCs in the presence of drug, but those tested did not react with ceftriaxone-treated RBCs. In addition to cefotetan-dependent antibodies, 19 (44%) and 14 (33%) of 43 sera contained drug-independent antibodies when tested with and without the presence of a polyethylene glycol potentiator, respectively. CONCLUSION Cefotetan is by far the most common cause of drug-induced IHA. All cefotetan antibodies and the single examples of cefoxitin and cefotaxime antibodies reacted with drug-coated RBCs, and most, in contrast to the reactions of antibodies to first-generation cephalosporins (e.g., cephalothin), also reacted with RBCs (not treated with drug) in the presence of the drug. Ceftriaxone antibodies reacted only by the latter mechanism. Drug-independent antibodies (i.e., those reacting without any drug being present) were detected in 33 to 44 percent of patients' sera containing cefotetan antibodies, depending on the sensitivity of the method used.