OBJECTIVE The Eph-ephrin system is a unique system that can induce multiple cellular responses such as cell migration, regulation of angiogenesis, and axonal guidance. Previously, the Eph-ephrin system was reported to regulate human extravillous trophoblast invasion. In this study, we examined the possible involvement of the Eph-ephrin system in the invasion of malignant gestational trophoblastic diseases using a human choriocarcinoma-derived cell line, JEG-3. METHODS The mRNA expression of class A Ephs and ephrins on JEG-3 cells was examined by reverse transcription-polymerase chain reaction. The effects of recombinant human Eph A1 (r-Eph A1) and r-ephrin A4 on the proliferation and invasion of JEG-3 cells were investigated by cell proliferation and Matrigel invasion assays. The alterations of integrin expression on JEG-3 cells in the presence of r-Eph A1 and r-ephrin A4 were investigated by flow cytometry. The induction of phosphorylation of focal adhesion kinase in JEG-3 cells by r-ephrin A4 was examined by Western blot analysis. RESULTS By reverse transcription-polymerase chain reaction, mRNAs of Eph A1, A2, and A4 and ephrin A1, A4, and A5 were detected on JEG-3 cells. In Matrigel invasion assay, both r-Eph A1 and r-ephrin A4 promoted the invasion of JEG-3 cells without affecting cell proliferation. During 24-hour culture with r-Eph A1 and r-ephrin A4, the increase in integrin α 5 expression on JEG-3 cells was observed by flow cytometry. Western blotting analysis showed that r-ephrin A4 induced dephosphorylation of focal adhesion kinase in JEG-3 cells. CONCLUSIONS These findings suggest that Eph-ephrin interaction plays some role in the regulation of choriocarcinoma invasion in cooperation with integrins.