Objective: Iodide transport across thyrocytes constitutes a critical step for thyroid hormone biosynthesis, mediated mainly by the basolateral sodium-iodide-symporter (NIS) and the apical anion exchanger pendrin (PDS). Both transmembrane proteins have been described as autoantigens in thyroid disease, yet the reports on autoantibody (aAb) prevalence and diagnostic usefulness are conflicting. Reasons for the inconclusive findings may be small study groups and principle differences in the technologies used. Design: We decided to re-evaluate this important issue by establishing novel non-radioactive tests using full-length antigens and comparable protocols, and analysing a large cohort of thyroid patients (n=323) and control samples (n=400). Methods: NIS and PDS were recombinantly expressed as fusion protein with firefly luciferase (Luc). Stably transfected HEK293 cells were used as reproducible source of the autoantigens. Results: Recombinant NIS-Luc showed iodide transport activity, indicating successful expression and correct processing. Commercial antibodies yielded dose-dependent responses in the newly established assays. Reproducibility of assay signals from patient sera was verified with respect to linearity, stability and absence of matrix effects. Prevalence of PDS-aAb was similar in thyroid patients and controls (7.7% versus 5.0%). NIS-aAb were more prevalent in patients (7.7% versus 1.8%), especially in Graves’ Disease (12.3%). Neither NIS-aAb nor PDS-aAb concentrations were related to TPO-aAb or TSH-receptor-aAb concentrations, or to serum zinc or selenium status. Conclusions: Our data highlight a potential relevance of autoimmunity against NIS for thyroid disease, whereas an assessment of PDS-aAb in thyroid patients seems not to be of diagnostic value (yet).