Methods of crossmatch testing prior to kidney transplantation are not standardized and there are limited large-scale data on the use and outcomes implications of crossmatch modality. Data describing the most sensitive crossmatch modality for crossmatch-negative kidney transplants were drawn from the Organ Procurement and Transplant Network Registry. Within the cohort transplanted in 1999-2005, we identified patient and transplant characteristics predictive of each testing modality by multivariate logistic regression. We assessed associations of crossmatch modality with rejection risk by logistic regression and with graft survival by Cox's hazards analysis. Among 230,995 transplants, use of flow cytometry with T-and B-lymphocytes (T&B FC) increased progressively in 1987-2005. Among the recent transplants performed in 1999-2005 (n=64,320), negative T&B FC crossmatch was associated with 15% lower relative risk of first-year acute rejection (adjusted HR 0.85, 95% CI 0.80-0.89) compared to negative T-antihuman-globulin and B-National Institutes of Health/Wash (T AHG &B) crossmatch. Five-year graft survival after transplant with negative T&B FC (82.6%) was modestly better than after negative T AHG &B (81.4%, P= 0.008) or T AHG crossmatch (81.1%, P 0.0001), but on adjusted analysis was significantly different only among recipients from deceased donors and patients aged > 60 years. Many subgroups for whom negative T&B FC crossmatch predicted lower rejection risk (Caucasians, deceased donor recipients, re-transplants) were not more likely to be crossmatched by this method. We conclude that current practice patterns have not aligned utilization of T&B FC crossmatch with associated benefits. Prospective evaluation of the relationship of crossmatch modality with outcomes is warranted.