Objective. To analyze the timing of Rhesus D (RhD) immunization in pregnancy and the consequences for the index pregnancy and for subsequent pregnancies to be able to optimize the design of antenatal screening and prevention programs. Design. Retrospective cohort study. Setting. Stockholm county, Sweden. Population. All RhD immunized pregnant women 1990-2008 before the introduction of routine antenatal anti-D prophylaxis. Methods. Data were collected from transfusion medicine registers and databases, medical records, the Swedish Medical Birth Register and the National Perinatal Quality Register and entered into a standardized database before analysis. Main outcome measures. The order of pregnancy and trimester when immunization occurred and treatment of hemolytic disease of the fetus and newborn. Results. A total of 290 RhD immunized women were included in the study. In 147/290 (51%) of the women, sensitization occurred with their first-born child and in 96/290 (33%) it occurred with their second-born child. Anti-D antibodies developed during the second or third trimester in 212/290 (73%) and in 61/290 (21%) at term or after delivery. In subsequent pregnancies 56% (144/259) of the neonates required treatment for hemolytic disease of the fetus and newborn. Conclusions. Based on our study, at least half of the cases could potentially have been avoided by routine antenatal anti-D prophylaxis in the beginning of the third trimester. To optimize the beneficial effects of new prevention programs, we propose providing anti-D prophylaxis in gestational week 28-30 selectively to all RhD-negative women with RhD-positive fetuses.Abbreviations: HDFN, hemolytic disease of the fetus and newborn; IgG, immunoglobulin G; IUT, intrauterine blood transfusion; RAADP, routine antenatal anti-D prophylaxis; RBC, red blood cell; RhD, Rhesus D.