Recipients of HLA-identical stem cell transplants have a poorer transplant outcome if the donor is female rather than male. We analyzed whether pregnancy primes for minor histocompatibility (H) antigens. Peripheral blood mononuclear cells (PBMCs) from healthy multiparous female blood donors were depleted for CD4+, CD14+, CD16+, and CD19+ cells, stained with minor H antigen-specific HLA-A2 tetramers, sorted by fluorescence-activated cell sorting, and tested for cytotoxic activity. Minor H antigens HY-, HA-1-, and HA-2-specific cytotoxic T cells (CD8+, CD45RA-) were present in PBMCs from 4 of 7 female donors up to 22 years after the last delivery. Interestingly, in 2 of the 4 cases microchimerism of the putative immunizing minor H antigen was observed. Thus, pregnancy can lead to alloimmune responses against the infant's paternal minor H antigens. The minor H antigen immunization status of female donors raises important questions for the clinical practice of stem cell transplantation.