BACKGROUND: Maternal thyroid dysfunction has been associated with a variety of adverse pregnancy outcomes. Laboratory measurement of thyroid function plays an important role in the assessment of maternal thyroid health. However, occult thyroid disease and physiologic changes associated with pregnancy can complicate interpretation of maternal thyroid function tests (TFTs). OBJECTIVE AND METHODS: To 1) establish the prevalence of laboratory evidence for autoimmune thyroid disease (AITD) in pregnant women; 2) establish gestational age-specific reference intervals for TFTs in women without AITD; and 3) examine the influence of reference intervals on the interpretation of TFT in pregnant women. Serum samples were collected from 2272 pregnant women, and TFT performed. Gestational age-specific reference intervals were determined in women without AITD, and then compared with the non-pregnant assay-specific reference intervals for interpretation of testing results. RESULTS: Thyroid peroxidase antibodies (TPO-Ab) and thyroglobulin antibodies (Tg-Ab) were positive in 10.4 and 15.7% of women respectively. TPO-Ab level was related to maternal age, but TPO-Ab status, Tg-Ab status, and Tg-Ab level were not. Women with TSH > 3.0 mIU/l were significantly more likely to be TPO-Ab positive. Gestational age-specific reference intervals for TFT were significantly different from non-pregnant normal reference intervals. Interpretation of TFT in pregnant women using non-pregnant reference intervals could potentially result in misclassification of a significant percentage of results (range: 5.6-18.3%). CONCLUSION: Laboratory evidence for thyroid dysfunction was common in this population of pregnant women. Accurate classification of TFT in pregnant women requires the use of gestational age-specific reference intervals.