Thyroid peroxidase (TPO) autoantibodies, a hallmark of human autoimmune thyroid disease, may have kappa or lambda light chains. Monoclonal human TPO autoantibodies with kappa light chains have previously been developed by cloning and expressing "combinatorial" libraries of immunoglobulin genes in bacteria. In the present study, an IgG1/lambda combinatorial library was generated from thyroid cDNA of a Graves' patient whose serum contained lambda TPO antibodies. Screening the bacteriophage library with 125I-TPO yielded one clone, TR1.41. The oligonucleotide sequence of TR1.41 was determined and the nature of its interaction with TPO was investigated. The affinity of TR1.41 for TPO is high (Kd approximately 10(-9) M), comparable to that of monoclonal kappa TPO autoantibodies derived from the same patient. The genes encoding the heavy and light chains of TR1.41 differ in a number of respects from the closest available germline genes. Such differences are consistent with somatic mutation in a high-affinity antibody. An important characteristic of TR1.41 is its interaction with the immunodominant domain on TPO recognized by approximately 80% of serum TPO autoantibodies. The frequency of TPO-specific F(ab) generated from the thyroid gland of patient TR was much lower for F(ab) with lambda light chains (1:150,000) than for F(ab) with kappa light chains (1:13,000). Despite this low frequency, the high affinity of TR1.41 and its recognition of the immunodominant region on TPO indicate that lambda autoantibodies of this type may represent an important constituent of the TPO autoantibody response in man. In conclusion, this is the first report on the molecular cloning and characterization of a thyroid autoantibody of lambda L chain type by the combinatorial library approach.