This study was designed to identify and characterize the allo- and autoantibodies induced following successful paternal lymphocyte immunization to prevent recurrent spontaneous abortion. Firstly the titers of maternal anti-paternal antibodies in women with successful pregnancies as determined by the flow cytometry crossmatch (FCXM) were highly variable; however, in all cases, the initial pre-immunization titers were negative and the post-immunization titers were positive by the FCXM in successfully treated women. Secondly, the specificities of maternal alloantibodies to paternal HLA antigens (immunogen) were evaluated. No all predicted antibodies to mismatched paternal HLA antigens were found by microlymphocytotoxicity (MCX) assays and the specificities varied. Thirdly, antibodies in post- but not preimmunization sera reacted with two lymphoid cell lines, SupT1 and SB; in addition, the rise and fall of the titers of these sera with paternal cells seemed to be reflected with the cell lines by the FCXM. Fourthly, autoantibodies to activated lymphocytes were detected and seemed to correlate with successful immunization since women who had another abortion following immunotherapy lacked these autoantibodies. These findings suggest that the antibody response following successful immunotherapy is complex and needs to be studied further to understand the mechanism of this treatment.