Infections have been implicated in the pathogenesis of a number of autoimmune diseases, and Yersinia enterocolitica (YE) might play a role in the development of autoimmune thyroid disease (AITD). Clinical evidence in support of this hypothesis has been inconclusive. We reasoned that looking earlier in the natural course of AITD might enhance chances of finding evidence for YE infection. Consequently, we determined seroreactivity against YE in subjects at risk of developing AITD, i.e. in 803 female relatives of AITD patients in self-proclaimed good health. As a comparison group we used 100 healthy women who participated in a program for reference values. IgG and IgA antibodies to virulence-associated outer membrane proteins (YOPs) of YE were measured by a specific assay. Serum thyroid peroxidase antibodies (TPO-Ab) as indicators of AITD were considered to be positive at levels of> 100 kU/l. The prevalence of YOP IgG-Ab was higher in AITD relatives than in controls (40.1% vs. 24%, P = 0.002), and the same was true for YOP IgA-Ab (22% vs. 13%, P < 0.05). Of the 803 AITD relatives, 44 had an increased or decreased plasma TSH, and 759 were euthyroid as evident from a normal TSH; the prevalence of YOP-Ab did not differ between these three subgroups. TPO-Ab were present in 10% of controls and in 27% of the AITD relatives (P < 0.001). The prevalence of TPO-Ab in the euthyroid AITD relatives was not different between YOP IgG-Ab positive and negative subjects (23.3% vs. 24.7%, NS), nor between YOP IgA-Ab positive and negative subjects (21.2% vs. 24.9%, NS). In conclusion, healthy female relatives of AITD patients have an increased prevalence of YOP antibodies, which, however, is not related to the higher prevalence of TPO antibodies in these subjects. The findings suggest a higher rate of persistent YE infection in AITD relatives. Susceptibility genes for AITD may also confer a risk for YE infection.