Tissue factor pathway inhibitor 2 (TFPI-2), a Kunitz-type proteinase inhibitor, might play an important role during placenta growth by regulating trophoblast invasion and differentiation. Many TFPI-2 transcripts have been detected in syncytiotrophoblast cells, but conflicting results have been reported concerning TFPI-2 synthesis by the cytotrophoblast. To address this issue, we developed a method to isolate pure preparations of human villous cytotrophoblast cells from normal term placentas, and the synthesis of tissue factor, TFPI-1, and TFPI-2 mRNAs was then evaluated. Cells were isolated by trypsin-DNase-EDTA digestion, followed by Percoll gradient separation and immunodepletion of human leukocyte antigen-positive cells. The quality of villous cytotrophoblast cells was verified by electron microscopy. Purity of cell preparations was assessed by labeling cells with GB25, a monoclonal antibody specific to villous trophoblast cells, and by checking the absence of contaminating cells using anti-CD9 antibody. The lack of hCG, CD32 mRNA, and tissue factor mRNA also indicated the absence of contaminating cells. Using competitive reverse transcription polymerase chain reaction, we showed that freshly isolated villous cytotrophoblast cells synthesized significant levels of TFPI-1 mRNA and larger amounts of TFPI-2 mRNA. TFPI-1 and TFPI-2 mRNA synthesis remained unchanged when cytotrophoblast cells were cultured in complete medium and evolved as a multinucleated syncytiotrophoblast. These results indicate that the villous cytotrophoblast and syncytiotrophoblast are both important sites of TFPI-2 synthesis in the human placenta. This study also indicates that tissue factor detection should be used systematically to check the purity of cytotrophoblast cell preparations because it allows detection of contamination by monocytes/macrophages and by syncytial fragments.