The purpose of this study was to determine the relationships between acute rejection, anti-major histocompatibility complex (MHC) class I and/or class II-reactive alloantibody production, and chronic rejection of renal allografts following kidney or simultaneous kidney-pancreas transplantation. Sera from 277 recipients were obtained pretransplant and between 1 month and 9.5 years post-transplant (mean 2.6 years). The presence of anti-MHC class I and class II alloantibodies was determined by flow cytometry using beads coated with purified MHC molecules. Eighteen percent of recipients had MHC-reactive alloantibodies detected only after transplantation by this method. The majority of these patients produced alloantibodies directed at MHC class II only (68%). The incidence of anti-MHC class II, but not anti-MHC class I, alloantibodies detected post-transplant increased as the number of previous acute rejection episodes increased (p = 0.03). Multivariate analysis demonstrated that detection of MHC class II-reactive, but not MHC class I-reactive, alloantibodies post-transplant was a significant risk factor for chronic allograft rejection, independent of acute allograft rejection. We conclude that post-transplant detectable MHC class II-reactive alloantibodies and previous acute rejection episodes are independent risk factors for chronic allograft rejection. Implementing new therapeutic strategies to curtail post-transplant alloantibody production, and avoidance of acute rejection episodes, may improve long-term graft survival by reducing the incidence of chronic allograft rejection.