Germ line insertion of a human endogenous retrovirus-like element (HERV-E.PTN) into the growth factor pleiotrophin (PTN) gene generated a phylogenetically new promoter driving the expression of functional HERV-PTN fusion transcripts. Here we show by in situ hybridization, that HERV-PTN fusion transcripts are expressed in malignant trophoblasts (i.e. choriocarcinoma) and in the proliferative and in the invasive trophoblasts of gestational trophoblastic tissue. Additionally, a 1.9 kb fragment of the HERV-derived PTN promoter was analysed which has strong activity when transiently transfected into choriocarcinoma JEG-3 cells in contrast to HeLa cells. Deletion of the retrovirally-derived promoter portion abolished its activity and an enhancer (+443 to +486) was identified in this region. Electrophoretic mobility shift and supershift experiments identified a Sp1 binding site in this enhancer and site specific mutation of this site abolished its activity in choriocarcinoma cells. Sp1 overexpression in Drosophila SL2 cells showed that the enhancer activity is mediated via Sp1 binding in vivo. Furthermore, mutation of the Sp1 binding site reduced the activity of a promoter test fragment in choriocarcinoma cells by 80%. Our result shows that a retroviral Sp1 binding site in the PTN promoter is important for the expression of growth factor pleiotrophin in human choriocarcinoma cells. Oncogene (2000) 19, 3988 - 3998.