Aprotinin is being used increasingly to limit cardiopulmonary bypass (CPB)-induced coagulation derangements. Since whole blood prothrombin time (PT) and activated partial thromboplastin time (APTT) assays are beneficial in the treatment of bleeding after CPB, we studied the potential effect of aprotinin on these whole blood assays. Blood specimens from 151 cardiac surgical patients were obtained in two phases: prior to heparin administration, before CPB, and subsequent to heparin neutralization after CPB. After collection, blood specimens were divided into two aliquots and mixed with either normal saline (NS) or aprotinin (A, 200 or 400 Kallikrein inhibiting units (KIU)/mL). Whole blood specimens were used to measure whole blood PT and APTT using CoaguChek® Plus instruments. Whole blood PT results were similar between normal saline (NS)- and aprotinin-spiked specimens before CPB (A, 12.9 ± 1.5 s; NS, 12.8 ± 1.5 s; P = 0.76) and after CPB (A, 17.5 ± 2.4 s; NS, 17.7 ± 2.4 s; P = 0.58). In contrast, whole blood APTT results were prolonged in aprotinin-spiked specimens prior to CPB (A, 63.3 ± 32.2 s; NS, 38.6 ± 16.3 s; P < 0.0001) and after CPB (A, 65.9 ± 23.7 s; NS, 45.7 ± 14.4 s; P < 0.0001). A dose-dependent prolongation of whole blood APTT by aprotinin was demonstrated by a greater mean difference in APTT (P = 0.0001) between specimens spiked with NS or 200 KIU (17.5 ± 12.2 s) vs 400 KIU (27.8 ± 21.5 s) of aprotinin. A greater mean difference in APTT values (P < 0.0001) between NS- and aprotinin-spiked specimens was demonstrated when comparing APTT measurements between specimens obtained from patients receiving (H) or not receiving (NH) heparin preoperatively (H, 36.5 ± 18 s; NH, 18.1 ± 21.9 s). Our data demonstrate that aprotinin-mediated prolongation of whole blood APTT is dose-dependent and enhanced by heparin. We conclude that when the whole blood APTT assay is used to assess heparin anticoagulant effect or coagulation factor levels after CPB, results should be interpreted with caution in the setting of concurrent aprotinin administration.