The horse early conception factor (ECF) test is designed for qualitative determination of the ECF glycoprotein in the mare that has conceived. The objectives of this study were to determine the performance of the horse ECF test for the detection of the non-pregnant mare, and to determine the agreement among subjects or "readers" regarding the interpretation of the test. Blood samples from 60 mares were collected on Days 0, 5, 8, 11 and 18 following ovulation. Pregnancy status diagnosed with the ECF test was compared (2 x 2 table) to pregnancy status diagnosed by palpation per rectum and ultrasound examination on Day 18 following ovulation. Three readers interpreted the ECF test outcome independently. Two laboratories independently interpreted the ECF test outcome from the same serum samples. Agreement was tested by kappa coefficient. Sensitivity, specificity, positive predictive value, negative predictive value, and accuracy ranged from 0.74 to 0.84, 0.14 to 0.33, 0.62 to 0.66, 0.33 to 0.44 and 0.57 to 0.60, respectively. Agreement between readers was substantial (0.60<kappa<0.77), but agreement among readers was fair (kappa=0.28). Agreement between laboratories was fair (kappa=0.28). We concluded that non-pregnancy status cannot be determined by the ECF test in the early pregnant mare with high accuracy.