Twenty cardiac surgical patients requiring cardiopulmonary bypass were enrolled in this study designed to evaluate the effect of aprotinin on activated clotting time (kaolin and celite), whole blood, and laboratory-based plasma (anti-Xa) heparin measurements. Whole blood heparin measurements were not different (p = 0.98) between aprotinin-treated (3.2 ± 2.8 U/mL) and control (3.2 ± 3.0 U/mL) specimens. Plasma anti-Xa heparin measurements were also not different (p = 0.95) between aprotinin-treated (2.7 ± 2.5 U/mL) and control (2.8 ± 2.5 U/mL) specimens. The relationship between whole blood (plasma equivalent) and plasma heparin measurements was similar (p0 = 0.1) in the presence (slope, 1.04; r2 =0.89) or absence (slope, 1.11; r2 = 0.89) of aprotinin. In contrast to weak correlations between celite (r = 0.50) or kaolin (r = 0.53) activated clotting time values, whole blood heparin measurements correlated well (r = 0.93) with plasma heparin measurements during cardiopulmonary bypass in the presence of aprotinin. These findings indicate that whole blood heparin measurements are unaffected by aprotinin and correlate well with plasma anti-Xa heparin measurements even in the presence of aprotinin. Therefore, the automated protamine titration assay can be used to monitor accurately heparin concentrations in patients receiving aprotinin.