Abstract: Molecular mimicry between the 65‐kDa isoform of glutamic acid decarboxylase (GAD65) and the protein 2C (P2C) of Coxsackie B4 virus (CBV) may initiate human type 1 diabetes. GAD65 contains a motif that has a 6‐amino acid identity with CBV‐P2C (PEVKEK), whereas the weakly autoantigenic isoform, GAD67, contains PEVKTK. A human‐derived monoclonal antibody (mAb) MICA3 reacts with a surface loop of GAD65 that includes PEVKEK, and mutagenic deletion of this loop was shown to reduce reactivity of GAD with the mAb by 70%. To establish that the PEVKEK motif on GAD65 contains a major epitope for diabetes sera and to identify the amino acids involved, mutants of nucleotides of GAD65 and GAD67 at sites in the PEVKEK motif were created and the expressed proteins used for radioimmunoprecipitation (RIP) tests with sera from patients with type 1 diabetes. A potent mouse mAb (GAD6) to GAD65, and a rabbit polyclonal antibody (AB108) to GAD67, were used to standardize the reactivity of the diabetes sera with the mutant molecules. Of 45 type 1 diabetes sera tested, 30 (67%) had an 80% or greater reduction of reactivity to GAD65_Δ258‐270 vs. intact GAD65. Various single‐surface amino acids in the PEVKEK epitope region of GAD65 were mutated, but most molecules carrying these mutations reacted similarly to the parent molecule. However after point mutation of the equivalent motif of GAD67 (PEVKTK to PEVKEK), there was an increase in the reactivity of 12 of 49 (24%) type 1 diabetes sera tested; 7 of 8 sera reactive with GAD67 showed increased reactivity with GAD67_T273E, and 5 previously negative sera gained reactivity with GAD67_T273E. Thus, the PEVKEK motif on GAD65 contributes to serologic reactivity of type 1 diabetes sera. This favors the hypothesis that CBV infection causes type 1 diabetes by the process of viral mimicry with cross‐reactivity to a critical epitope of GAD65.