Autoimmune thyroid disease (AITD) is often familial and serological HLA disease associations have been described in many different populations. However, such HLA disease associations are weak and the precise molecular contribution of HLA antigens to thyroid disease susceptibility remains unknown. Much of the data available are cross-sectional and few studies have explored familial inheritance of AITD at the molecular level. We have, therefore, examined the inheritance of AITD in multiplex and multi-generational families using restriction fragment length polymorphism (RFLP) analysis of DNA digested with the restriction enzyme BamHl and probed with a full length human HLA-DQ beta cDNA probe. Thirty seven subjects in 7 informative families were available for study. Eleven subjects had Graves' disease and 4 were diagnosed as having Hashimoto's thyroiditis. Segregation of polymorphic fragments enabled genotyping of each individual to produce fully informative families. LOD scores were computed, using the LIPED program, for dominant and recessive models of inheritance, for recombination fractions of 0.01 to 0.5 for each sex, and for penetrances of 0.1 to 1.0. The results showed that maximum LOD scores were negative for all of the inheritance models tested. If the primary locus for AITD were in the HLA region, LOD scores would be highly positive. These data. therefore, provide strong evidence against a disease locus for AITD in linkage disequilibrium with the HLA-DQ beta locus.