BACKGROUND: Tissue factor (TF) pathway inhibitor-1 (TFPI) is expressed in several malignant tissues- and cell lines and we recently reported that it possesses anti-tumor effects in breast cancer cells, indicating a biological role of TFPI in cancer. The two main splice variants of TFPI; TFPIa and TFPIß, are both able to inhibit TF-factor VIIa (FVIIa) activity in normal cells, but only TFPIa circulates in plasma. The functional importance of TFPIß is therefore largely unknown, especially in cancer cells. We aimed to characterize the expression and function of TFPIa, TFPIß, and TF in a panel of tumor derived breast cancer cell lines in comparison to normal endothelial cells. METHODS: TFPIa, TFPIß, and TF mRNA and protein measurements were conducted using qRT-PCR and ELISA, respectively. Cell-associated TFPI was detected after phosphatidylinositol-phospholipase C (PI-PLC) and heparin treatment by flow cytometry, immunofluorescence, and Western blotting. The potential anticoagulant activity of cell surface TFPI was determined in a factor Xa activity assay. RESULTS: The expression of both isoforms of TFPI varied considerably among the breast cancer cell lines tested, from no expression in Sum149 cells to levels above or in the same range as normal endothelial cells in Sum102 and MDA-MB-231 cells. PI-PLC treatment released both TFPIa and TFPIß from the breast cancer cell membrane and increased TF activity on the cell surface, showing TF-FVIIa inhibitory activity of the glycosylphosphatidylinositol- (GPI-) anchored TFPI. Heparin treatment released TFPIa without decreasing the cell surface levels, thus indicating the presence of intracellular storage pools of TFPIa in the breast cancer cells. CONCLUSION: GPI-attached TFPI located at the surface of breast cancer cells inhibited TF activity and could possibly reduce TF signaling and breast cancer cell growth locally, indicating a therapeutic potential of the TFPIß isoform.