Despite more than two decades of use, the optimal maintenance dose of tacrolimus for kidney transplant recipients is unknown. We hypothesized that HLA class II donor-specific antibody (DSA) development correlates with tacrolimus trough levels and the recipient's individualized alloimmune risk determined by HLA-DR/DQ epitope mismatch. A cohort of 596 renal transplant recipients with 50,011 serial tacrolimus trough levels had HLA-DR/DQ eplet mismatch determined using HLAMatchmaker software. We analyzed the frequency of tacrolimus trough levels below a series of thresholds <6 ng/ml and the mean tacrolimus levels before DSA development in the context of HLA-DR/DQ eplet mismatch. HLA-DR/DQ eplet mismatch was a significant multivariate predictor of DSA development. Recipients treated with a cyclosporin regimen had a 2.7-fold higher incidence of DSA development than recipients on a tacrolimus regimen. Recipients treated with tacrolimus who developed HLA-DR/DQ DSA had a higher proportion of tacrolimus trough levels <5 ng/ml, which continued to be significant after adjustment for HLA-DR/DQ eplet mismatch. Mean tacrolimus trough levels in the 6 months before DSA development were significantly lower than the levels >6 months before DSA development in the same patients. Recipients with a high-risk HLA eplet mismatch score were less likely to tolerate low tacrolimus levels without developing DSA. We conclude that HLA-DR/DQ eplet mismatch and tacrolimus trough levels are independent predictors of DSA development. Recipients with high HLA alloimmune risk should not target tacrolimus levels <5 ng/ml unless essential, and monitoring for DSA may be advisable in this setting.