The sensitivity to heparin during cardiopulmonary bypass (CPB) is determined by patient-specific characteristics and is assessed by the whole blood activated clotting time (ACT). We aimed to examine reliability measures between two different ACT monitors using Bland-Altman analysis: bias should not exceed 50 ± 50 seconds for measurements performed during CPB or 10 ± 10 seconds before and after CPB. The ACT response should be linear in relation to the concentration of heparin in plasma. Twenty patients (n = 20) aged 20-80 years and admitted for coronary artery bypass surgery were enrolled to this clinical observational study. ACT values and antifactor Xa were sampled: 1) before induction of anesthesia, 2) after heparin bolus, 3) during CPB at the start of rewarming, 4) at weaning from CPB, and 5) after heparin reversal. The evaluation comprised the Hemostasis Management System Plus™ (HMS, Medtronic Inc., Minneapolis, MN) and i-STAT™ (Abbott, Point of Care Inc., Princeton, NJ). Bias for the HMS Plus™ vs. i-STAT™ was +105 ± 119 seconds for measurements during CPB and +2.8 ± 11.7 seconds before and after CPB. Associated limits of agreement for the observed bias were ±235 and ±23 seconds, respectively. Inter-device correlation of ACT values was .46 (p < .001) during CPB; otherwise .48 (p = .02). Both devices produced ACT values unrelated (<10%) to the measured heparin concentration. The use of multivariable regression analysis demonstrated an independent association between the ACT measurement and hematocrit, however, not with the plasma concentration of heparin. ACT monitors demonstrate unacceptable bias differences, combined with wide limits of agreement. The ACT response correlated with hematocrit, but not with the actual heparin concentration.