After liver surgery, two patients developed unexplainable prolonged prothrombin times (PT) that were not associated with bleeding tendency. The substitution of rabbit thromboplastin for either human or monkey thromboplastin in performing PT tests resulted in a normal clotting time. Tissue factor (TF) procoagulant activity assays and an immunoblotting analysis showed that these patients had developed IgG lambda-type immediate anticoagulants directed against both rabbit and bovine TF that did not crossreact with human or monkey TF. In a chromogenic assay, the patient IgG caused a decrease in both the Km and the Vmax of the factor X activation by rabbit TF-factor VIIa complex. The lack of reactivity of the patient IgG with human TF presumably explained why there was no clinical bleeding. Both patients had been treated earlier with a topical hemostatic agent prepared from bovine corium, microfibrillar collagen hemostat, while undergoing previous surgery. In an immunoblotting analysis, the patient IgG stained a 42-Kd band in the Triton extract of the collagen preparation under either reducing or nonreducing conditions. The Triton extract of the collagen preparation blocked the binding of the patient IgG to bovine TF. Thus, it is suggested that the iatrogenic immunization by intraoperative exposure of bovine TF retained in the collagen preparation may be responsible for the development of anti-TF antibodies in these patients. The anti-TF antibodies resulted in a clinical error in the evaluation of coagulation status after the use of rabbit thromboplastin.