This study was conducted to investigate whether antiphospholipid antibodies (APA) can interfere with the phospholipid-dependent inhibition of coagulation exerted by tissue factor pathway inhibitor (TFPI). Eleven patients with APA and eleven healthy controls matched for age and gender were enrolled. Blood samples were drawn before and 5 minutes after an intravenous injection of unfractionated heparin 5000 IE, which is known to cause TFPI release in healthy individuals. The preheparin samples showed significantly higher TFPI free antigen levels in the APA positive patients than in the controls (21.7 vs. 14.2 ng/ml, p = 0.03). TFPI activity as measured in a chromogenic substrate assay also was higher in patients, but this difference was not statistically significant (1.13 vs. 1.01 U/ml, p = 0.2). The TFPI levels showed a considerable rise in both patients and controls after heparin injection. In both assays, the postheparin levels were significantly higher in patients than in controls (TFPI antigen: 179 vs. 153 ng/ml, p = 0.05; TFPI activity: 3.26 vs. 2.51 U/ml, p = 0.03). A modified diluted prothrombin time assay (dPT) was used to measure TFPI anticoagulant activity. In this assay, samples from the patients with the strongest effect of lupus anticoagulants (LAs) on preheparin coagulation times showed little or no increase after heparin injection. This result may reflect an inhibition of TFPI anticoagulant activity by strong LAs. In conclusion, we have found that patients with APA have higher TFPI amidolytic activity/antigen level both before and after heparin stimulation of TFPI release. These observations do not explain the higher thrombotic risk in these patients but may reflect an upregulated tissue factor activity, which has been demonstrated in these patients. TFPI anticoagulant activity, however, as measured in a dPT assay, may be inhibited by strong LAs.