Since immune memory in Rh(D)-negative isoimmunized subjects remains through life, even in the absence of measurable anti-Rh(D), we investigated the transformation of lymphocytes from such donors by Rh(D) antigen. The time lapse from the last stimulus was up to 13 years. Mononuclear cells from immunized women were stimulated by Rh(D)-positive erythrocyte stroma. Maximum transformation was observed on the sixth day of culture, with a stroma protein concentration of 8 micrograms/mL of culture medium. The stimulation index (SI) in cells from 11 immunized women was 6.8 +/- 3.1 (mean +/- SD), with a range from 3.1 to 15.0. In five different sets of control cultures, the SI ranged from 0.9 +/- 0.2 to 1.3 +/- 0.4. There was no overlap between stimulated and control cultures. No anti-D could be demonstrated in the serum of four of the 11 immunized cases studied. Also, transformation was observed in mononuclear cells from Rh(D)-negative immunized women with Rh(D)-positive erythrocytes. The findings demonstrate that lymphocytes from isoimmunized Rh(D)-negative subjects maintain the immune memory and are transformed in vitro by the Rh(D) isoantigen.