To evaluate the influence of a positive B cell flow cytometry crossmatch (FCXM) on transplant outcome, we retrospectively performed B cell FCXMs for 431 consecutive cadaver renal transplant recipients using the two most current pretransplant sera. All transplant recipients had a negative lymphocytotoxic antiglobulin T cell XM and a negative (< or = 10 channel shift) T cell FCXM. B cell FCXMs were performed using a two-color technique to identify binding of IgG antibody to donor lymph node B lymphocytes stained for CD20. The incidence and causes of graft failure posttransplant were determined by requesting this information from recipient transplant centers. Transplants that failed due to nonimmunological causes (n = 54, 13%) were excluded from the analysis. Minimum follow-up was 12 months. We found no difference in graft survival at one year for transplants where the B cell FCXM was positive in the range of 11 to 50 channel shift (n = 201) compared with those with a negative (< or = 10 channel shift) B cell FCXM (n = 141)--i.e., 90% vs. 91%, P = NS. However, when the positivity in the B cell FCXM was > 50 channel shift (n = 35), significantly fewer grafts survived at one year, compared with those where the channel shift was < or = 50 (n = 342), 63% vs. 91%, P < 0.001. This was true for first transplants as well as regrafts and for transplants performed with a positive as well as a negative standard B cell XM. The detrimental effect of a positive B cell FCXM was seen for sensitized (PRA > 10% at the time of transplant) as well as nonsensitized patients. However, this effect was observed only when the donor had at least a one-DR mismatch. We conclude that a strongly positive B cell flow cytometry crossmatch identifies patients who are at risk for graft loss. Since the risk appears to be only when there is a DR mismatch, the data suggest that the B cell-specific IgG antibody detected by flow cytometry may be specific for the mismatched MHC class II antigens of the donor.