Glutamic acid decarboxylase-65 (GAD65), the enzyme that catalyzes the formation of gamma-aminobutyric acid (GABA), is the major autoantigen in both type 1 (insulin-dependent) diabetes and stiff-man syndrome (SMS). The observation that GAD65 autoantibodies may be present for years before the clinical onset of diabetes raises the question of when GAD65 is available to initiate an immune response to allow the formation of autoantibodies. In order to address this question it will be necessary to measure GAD65 in tissue, cells, and plasma.A radioimmunoassay (RIA) was developed for GAD65 based on the use of a polyclonal rabbit antiserum directed to the N-terminus of GAD65.Using the GAD65 RIA, we have determined the GAD65 content in a human GAD65 gene transfected cell line and in beta-cell preparations from different species. The assay detects an increase of immunoreactive GAD65 after glucose-stimulation and GAD65 that is discharged from rat beta cells after their exposure to the diabetogenic agent streptozotocin. It also measures good recovery of GAD65 added to human plasma samples.The GAD65 RIA makes it possible to determine both cellular and extracellular levels of GAD65; this might be useful in investigating the mechanisms leading to the formation of GAD65 autoantibodies in type 1 diabetes and SMS patients.