Background The line of differentiation in synovial sarcoma still remains controversial. Thus far, only a few human synovial sarcoma cell lines have been described. However, their morphologic characteristics have not been fully established. Experimental design We established a new synovial sarcoma cell line (HS-SY-II) from pleural effusion with lung metastasis in a typical example of the monophasic spindle cell type. The HS-SY-II cells, in vitro and in vivo, were examined by light microscopy, immunohistochemistry, electron microscopy, and cytogenetics. Results The HS-SY-II cells showed a hypertriploid karyotype with complex chromosome abnormalities including pathognomonic t(X;18)(p11;q11), and have been stably maintained for more than 40 months in vitro, showing rather small spindle or polygonal shape without conspicuous pleomorphism. Histologic features of initially and serially transplanted tumors in nude mice were essentially the same as those of the original sarcoma, corresponding to the monophasic spindle cell variant with a prominent palisading pattern and calcified foci in parts. The HS-SY-II cells in vitro and in vivo similarly expressed vimentin and cytokeratin by immunohistochemistry, and also exhibit the same ultrastructural features such as irregularly shaped nuclei with prominent nucleoli, many paranuclearly running intermediate filaments, and filopodia-like processes. Conclusions This HS-SY-II cell line retaining the distinct morphological characteristics as the monophasic spindle cell type of synovial sarcoma therefore will be extremely useful for various pathomorphologic investigations on synovial sarcoma.