The ability to detect conception and/or conception failure in cattle would be beneficial to producers in formulating reproductive management plans. A new diagnostic test, the early conception factor (ECF) test, has been developed forthis application yetthe accuracy of this test has not been adequately determined. The objectives of this study were to evaluate the effectiveness of the ECF test for detecting the nonpregnant cow, and to compare the reliability of serum versus milk ECF tests relative to actual pregnancy rates. In Trial 1, Holstein heifers were synchronized, the animals were bred (timed-AI), and serum ECF tests were performed 72 h later. Heifers exhibiting a negative ECF test after AI were re-synchronized, bred again, and re-tested for ECF for up to three services. Relative to actual pregnancy rates, a negative ECF test was correct (i.e., true negative) 38.5% of the time over the three services. In Trial II, Holstein heifers were bred (AI) after observed estrus and serum ECF tests conducted between Days 1 and 3 and Days 7 and 9 after AI. In this trial, only 44.4% and 55.6% of the confirmed nonpregnant heifers were identified correctly by serum ECF analysis at Days 1 to 3 and Days 7 to 9 post-AI respectively. In Trial III, 40 lactating cows were synchronized, the animals were bred (AI), and serum and milk ECF tests were performed on Days 3, 9, 15, 21 and 30 after AI. Pregnancy diagnosis (ultrasound on Day 30 and palpation on Day 51) confirmed that 50% of the cows were pregnant to AI, while serum and milk ECF analysis indicated a 100% and 37.5% predicted pregnancy rate, respectively, at 30 d post-AI. Moreover, results of the serum and milk ECF tests disagreed with one another 36.9% of the time overall, while agreement between ECF and actual pregnancy rates were 50.6% and 45.6% for milk and serum respectively. Additionally in Trial III, a negative ECF result only identified 5% and 28.8% of nonpregnant cows overall for serum and milk tests respectively (i.e., true negatives), with a high incidence of false positive ECF results noted (47.5% and 31.3% for serum and milk, respectively). Collectively, these data indicate that the current ECF test cannot accurately identify the nonpregnant cow with the precision needed by the dairy producer.