Thyroid-stimulating hormone (TSH) is the main regulator of thyroid gland function, and elevated TSH in humans is known to be associated with infertility. The aim was to establish an ELISA for measurement of bovine TSH and to compare TSH serum concentrations of early pregnant (n=19) and non-pregnant (n=19) Holstein Friesian heifers. For the TSH ELISA, an antibody targeted against bovine TSH (anti-bovine TSH, 1:10 pre-diluted, AFP-642482Rb, obtained from the National Hormone & Peptide Program (NHPP), NIDDK and Dr. Parlow) was diluted (1:2500). The applied standard curve ranged from 0.2–100 ng/ml bovine TSH (AFP-8755B, obtained from the NHPP), and biotin-labeled TSH was used to detect binding with subsequent addition of streptavidin horseradish peroxidase solution. The intraassay CV was determined by measuring one bovine sample 20 times. Serum samples of 19 matched pairs were used to compare pregnant and non-pregnant heifers. Blood was taken from the jugular vein on the Day of ovulation (defined by ultrasound; Day 0) and Days 14 and 18. Pregnancy status was defined as follows: pregnant = progesterone [ng/ml] >2.0 on Day 18 and successful conceptus collection or detectable embryonic heartbeat in ultrasound investigations on Day 42; non-pregnant = progesterone <1.0 on Day 18 and no trophoblast tissue in uterine flushing. The intraassay CV was 15.4 and the lowest detection limit was 0.6 ng/ml. Serum concentrations of TSH [ng/ml] on Day 0 were higher in non-pregnant (2.72±0.96) compared to pregnant (2.08±0.90) heifers (P<0.01). These results indicate that TSH as an important hormone reflecting the metabolic status around ovulation might have predictive potential for pregnancy outcome in heifers.