BACKGROUND The aim of this study was to determine the frequency and origin of additional alloantibodies directed against red cells (RBCs) after intrauterine transfusion (IUT). METHODS Between March 1987 and December 1992, fetuses with severe hemolytic disease (n = 91) received a total of 280 ultrasound-guided IUTs of RBCs from unrelated donors. The specificity of alloantibodies to RBCs in maternal serum was determined both before and after each IUT. If additional alloantibodies directed against RBCs were detected, their origin was determined by phenotyping the fetal, donor, and paternal RBCs for each particular antigen. The study included a control group of 69 pregnant women who underwent either amniocentesis or fetal blood sampling. RESULTS Production of additional alloantibodies directed against RBC antigens was detected in 24 women (26%). The source of the immunizing antigen, either donor or fetus, was identified in 14 patients. The additional alloantibodies were directed against fetal antigens in 11 women and against donor antigens in 3. One additional alloantibody directed against donor antigen clearly reduced the survival of donor RBCs. The fetus and the donor shared the immunizing antigen in four cases, and in one case, the antibody occurred naturally. In five cases, the source of the immunizing antigen was not determined. In the control group, additional antibodies were detected in two patients. CONCLUSIONS IUT therapy is associated with a high incidence of additional alloantibodies. In the majority of patients, the use of maternal RBCs for IUT would not have prevented additional formation of alloantibody to RBCs.