Background: Before the onset of fetal thyroid hormone production, the transplacental delivery of maternal thyroid hormones is necessary for embryonic and fetal development. Therefore, the adaptation of maternal thyroid hormone metabolism may be important for pregnancy success and embryo survival. The aims of this study were to determine the thyroid hormone levels during the early peri-implantation period until day 18 and on the day of ovulation, to determine whether pregnancy success is dependent on a "normothyroid status" and to determine whether physiological adaptations in maternal thyroid hormone metabolism occur, which may be necessary to provide sufficient amounts of biologically active T-3 to support early pregnancy. Therefore, blood samples obtained on the day of ovulation (day 0) and days 14 and 18 of the Holstein-Friesian heifers (n = 10) during the respective pregnant, non-pregnant and negative control cycles were analyzed for thyroid-stimulating-hormone (TSH), thyroxine (T-4) and triiodothyronine (T-3). Liver biopsies (day 18) from pregnant and respective non-pregnant heifers were analyzed for mRNA expression of the most abundant hepatic thyroid hormone deiodinase (DIO1) by real time qPCR. Results: Although liver DIO1 mRNA expression did not differ between the pregnant and non-pregnant heifers on day 18, the serum concentrations of TSH and T-3 on day 18 were higher in non-pregnant heifers compared to pregnant heifers (P < 0.05). Moreover, T-3 decreased between day 0 and 18 in pregnant heifers (P < 0.001). Conclusions: In conclusion, no associations between thyroid hormone patterns on day 18 and pregnancy success were detected. During the early peri-implantation period, TSH and T-3 may be affected by the pregnancy status because both TSH and T-3 were lower on day 18 in pregnant heifers compared to non-pregnant dairy heifers. In further studies, the thyroid hormone axis should be evaluated throughout the entire gestation to confirm these data and identify other possible effects of pregnancy on the thyroid hormone axis in cattle.