Using serum from a prediabetic patient as a probe, we screened 0.5 x 10(6) recombinants from a rat islet lambda gt11 expression library. One plaque-producing antigen reactive with this prediabetic serum was identified, subcloned, and sequenced. Analysis of the sequence reveals that the clone encodes a 136-amino acid fragment of carboxypeptidase-H (enkephalin convertase). Carboxypeptidase-H is a molecule expressed within islet secretory granules and neurendocrine cells. The patient whose antibodies recognize this recombinant molecule (termed DG-1) was negative for anti-DG-1 antibodies in 1984, developed the antibodies by 1986, and remained positive until the development of diabetes in 1988. To date, serum from 5 of 20 cytoplasmic islet cell antibody-positive relatives reacted with the expressed protein, while none of 14 control sera reacted. On Western blotting, the initial patient's serum used for the screening reacts with a 52-kDa antigen corresponding to the mol wt of the membrane form of carboxypeptidase-N. The current study has identified carboxypeptidase-H as an autoantigen recognized by serum of pretype I diabetes, and the methodology used should aid in identification of additional autoantigens associated with type I diabetes.