Screening 10(6) plaque-forming units from a lambda gt11 human thyroid cDNA library with a pool of 10 sera from patients with Hashimoto's thyroiditis resulted in the isolation of a clone, D1, of 292 basepairs with an open reading frame of 97 amino acids which did not share significant similarity with any known protein. Rescreening the library using D1 as a probe led to the full-length cDNA being cloned and sequenced which has the potential to encode a 572-amino acid polypeptide. This would correspond to a 63-kDa nonglycosylated protein which is probably membrane bound. The recombinant fusion protein from the original D1 clone was recognized in dot blot or Western blot assays by 4 of 19 patients with Hashimoto's thyroiditis, 3 of 9 patients with Graves' disease, and 1 of 6 patients with idiopathic myxedema; all other sera (12), including that from normal subjects, were negative. Clone affinity-purified autoantibodies bound to a protein of 64 kDa in a Western blot of human thyroid tissue. Using D1 as a probe in a Northern blot revealed 3.9-kilobase (kb) transcripts in poly(A)+ RNA from normal human thyroid and extraocular muscle, but not skeletal muscle. The dog equivalent of D1 cDNA was isolated by screening 10(6) plaque-forming units from a dog thyroid cDNA library using clone D1. A fragment of dog cDNA was, in turn, used as a probe in a Northern blot of dog poly(A)+ RNAs from thyroid, brain, lung, heart, liver, kidney, spleen, and stomach; a faint signal at 3.9 kb was observed in all tissues except the thyroid, which displayed a strong transcript of 2.6 kb. It is concluded that the D1 cDNA clone encodes an autoantigen shared by the thyroid and eye muscle. Its possible relevance to autoimmune ophthalmopathy is discussed.