The study of the mammalian sex-determining pathway has been hampered by the lack of cell culture systems to investigate the underlying molecular relationships between sexdetermining genes. Recent approaches using high-throughput genome-wide studies have revealed a number of sexually dimorphic genes expressed in the developing mouse gonad. Here, we investigated a human testicular cell line in terms of its expression of known sex-determining genes and newly identified candidates. The human embryonal carcinoma cell line NT2/D1 was screened for the expression of 46 genes with known or potential roles in the sex-determining and differentiation pathway. Forty genes tested were expressed in NT2/D1 cells including the testis-determining genes SRY , SOX9 , SF-1 , DHH and FGF9 . Genes not ex-pressed included WT1 , DAX1 and the ovary-specific genes FOXL2 and WNT4 . Cell-specific markers demonstrate that NT2/D1 cells reflect a number of cell types in the gonad including Sertoli, Leydig and germ cells. Our results suggest that male pathways initiated by SRY, SOX9 and SF-1 remain intact in these cells. Lack of expression of ovary-specific genes is consistent with a commitment of these cells to the male lineage. Manipulation of gene expression in this cell line could be an important new in vitro tool for the discovery of new human sex-determining genes.