The autoimmune thyroid diseases (AITDs) are female-predominant diseases with a ratio of approximately seven females to each male. X chromosome inactivation (XCI), an epigenetic phenomenon, has been suggested to be skewed in many such female patients with AITD. We analyzed female genomic DNA from 87 patients with Graves' disease (GD), 47 patients with Hashimoto's thyroiditis (HT), and 69 healthy controls. Using an XCI assay based on Hpa II digestion and PCR and DNA sequencing, we found skewed heterozygous XCI (>or=80%) in 20 of 70 GD patients (28.6%) and 11 of 43 HT patients (25.6%), giving a total of 31 of 113 AITD patients (27.4%) with skewed XCI. In contrast, only 5 of 58 healthy controls had skewed XCI (8.6%). Statistical analysis confirmed that XCI skewing was significantly associated with AITD (P = 0.004, OR = 4.0), demonstrating that the degree of XCI is an important contributor to the increased risk of females in developing AITD.