Recent studies of the anticoagulant activities of the tissue factor (TF) pathway inhibitor (TFPI) isoforms, TFPIα and TFPIβ, have provided new insight into the biochemical and physiological mechanisms that underlie bleeding and clotting disorders. TFPIα and TFPIβ have tissue-specific expression patterns and anticoagulant activities. An alternative splicing event in the 5' untranslated region allows for translational regulation of TFPIβ expression. TFPIα has 3 Kunitz-type inhibitor domains (K1, K2, K3) and a basic C terminus, whereas TFPIβ has the K1 and K2 domains attached to a glycosylphosphatidyl inositol-anchored C terminus. TFPIα is the only isoform present in platelets, whereas endothelial cells produce both isoforms, secreting TFPIα and expressing TFPIβ on the cell surface. TFPIα and TFPIβ inhibit both TF-factor VIIa-dependent factor Xa (FXa) generation and free FXa. Protein S enhances FXa inhibition by TFPIα. TFPIα produces isoform-specific inhibition of prothrombinase during the initiation of coagulation, an anticoagulant activity that requires an exosite interaction between its basic C terminus and an acidic region in the factor Va B domain. Platelet TFPIα may be optimally localized to dampen initial thrombin generation. Similarly, endothelial TFPIβ may be optimally localized to inhibit processes that occur when endothelial TF is present, such as during the inflammatory response.