We analyzed the genetic regulation of spontaneous autoimune thyroiditis (SAT) by means of crosses between Obese strain (OS) chickens with the disease. and healthy inbred chicken of line CB. Mononuclear cell infiltration of the thyroid was used as a criterion for the disease. We confirmed the existence of one recessive gene, regulating the susceptibility of the thyroid gland to autoimmune attack. From the frequency of progeny with the thyroid infiltration in backcross and F2 generations, we presume the existence of one or two additional dominant genes coding for abnormal reactivity of the immune system. The total number of genes regulating SAT is therefore a maximum of three. We attempted to identify disease-specific transcripts responsible for the initiation of the disease using suppression subtractive hybridization of RNA prepared from OS and CB thyroid lobes, obtained from 3-day-old chicks. From forward and reverse subtractions. we recovered a fragment mixture in the range of 300 bp to 1.5 kb. In total. 768 clones were screened and 9 were sequenced. Four of them represent unknown sequences. Two, specific for OS thyroid, correspond to envelope genes of avian endogenous viruses (ev)-1, -3 and -6. The expressed product of an em, gene could be iodinated in the thyroid gland and become involved in thyroglobulin metabolism. This may be another possible mechanism driving SAT, as iodine modulates SAT in the chicken. Further experiments will be required to prove this hypothesis. Two thyroid-specific clones had significant alignment to human thyroglobulin, and one clone to human coatomer protein. We analyzed, by RFLP and RNA dot blots, cosegregation between clones for the em, gene of endogenous viruses as the most promising candidate for involvement in driving SAT, we did not find differences at the DNA and RNA levels. We can possibly therefore rule out a simple involvement of env genes with the initiation of disease.