Autoantibodies to thyroid peroxidase (TPO) are characteristic of thyroid inflammation in autoimmune thyroid disease. We have used the phage display, H and L chain combinatorial cDNA library approach to clone, from thyroid-infiltrating B cells, six new human Fab autoantibodies with high affinities (approximately 10(-10) M) for TPO. This library, in the pComb3 vector, was screened with viable, stably transfected Chinese hamster ovary cells expressing human TPO on their surface. The H and L chain genes in the six TPO-specific Fab were similar, but not identical, to those encoding Fab previously isolated from the same library by screening bacteriophage plaques in the Immunozap vector with purified TPO. The TPO-specific VK genes isolated with the phage display system are closer to germline than those obtained with Immunozap. Essentially all the V kappa isolated with pComb3 were 99 or 100% homologous with the germ-line genes KL012 and A3 that also code for low affinity systemic autoantibodies. There are two important implications of the study. First, the phage display system can be used with impure Ag to generate high affinity autoantibodies. This finding opens the way to cloning autoantibodies against other autoantigens, not previously possible with the bacteriophage lambda approach because of the lack of purified Ag. Second, germ-line L chain genes can code for very high affinity antibodies.