Autoantibodies against amino acid 200-239 (p200) in the predicted leucine zipper region of the Ro52 protein are associated with congenital heart block, a potentially fatal condition that may affect fetuses of women with Ro52 autoantibodies. To allow detailed studies of the antibodies associated with congenital heart block, B-cell derived combinatorial antibody libraries from patients were screened for Ro52 and p200 specific antibody clones. Two human monoclonal anti-p200 antibody fragments, S3A8 and M4H1, were isolated and analysed with regard to VHand VL gene utilization, somatic mutations and binding properties. Both identified clones recognized recombinant and native intact Ro52, and reacted only with p200 in a set of related Ro52 peptides. The specificity and affinity was confirmed by biosensor measurements. Structural analysis of overlapping peptides revealed increased helicity in the p200 peptide compared to non-recognized peptides, indicating epitope conformation as essential for antibody binding. Both monoclonals produced punctate nuclear and diffuse cytoplasmic staining in human and mouse cell lines. The identified antibodies, which react specifically with the leucine zipper structure of Ro52, will be valuable in further exploration of the mechanisms operating during development of Ro52 antibody-associated congenital heart block.