The treatment of maternal hypothyroidism presents clinicians with a unique challenge, because dosing regimens previously developed and validated for nonpregnant women cannot be easily extrapolated to dosing in pregnancy. Thyroid hormone requirement increases by 20% to 40% early during pregnancy, persisting throughout gestation. Accordingly, women with treated hypothyroidism need to increase their levothyroxine dose to prevent maternal hypothyroidism and the associated impaired cognitive development and increased fetal mortality. We investigated the pharmacokinetic properties of levothyroxine during pregnancy through the use of a novel, traceable form of levothyroxine. The objective was to conduct a longitudinal study to determine whether levothyroxine pharmacokinetics differ in the pregnant versus nonpregnant state. We used a unique C-levothyroxine-tracer method to distinguish between endogenous and exogenous levothyroxine and studied the pharmacokinetics of a single oral dose of levothyroxine using tandem mass spectrometry. Moreover, we were able to detect single dose amounts of the drug, in picogram/mL concentrations. The area under the curve was 23.0 ng*h/mL in pregnancy and 14.8 ng*h/mL in nonpregnant women (P < 0.03) with median serum half-lives of 32.1 hours and 24.1 hours, respectively (P < 0.04). Further research involves the measurement of free thyroxine on these samples using tandem mass spectrometry. Future work should focus on the mechanisms responsible for the gestational differences in pharmacokinetics and whether these should necessitate dose schedule changes in pregnancy.