The tumor suppressor retinoblastoma gene (RB) is a well-known regulator of cell cycle progression, differentiation and apoptosis. Inactivation of this gene is closely associated with the development of many human malignancies. Previous studies of testicular germ cell tumors (TGCTs) revealed lower levels of RB mRNA and protein compared with normal testis tissue. In this study, we examined the mechanism behind the reduced expression of RB mRNA and protein in TGCTs. The mRNA and protein expression were down-regulated in JKT-1 human testicular seminoma cells, compared with other cell lines. Promoter activity from a construct without a hGABP/E4TF1-binding site was markedly reduced in A549 human lung adenocarcinoma cells, but not in JKT-1. Furthermore, promoter activity from a plasmid carrying a point mutation at the hGABP/E4TF1 site was reduced in A549 cells, but not altered in JKT-1 cells. These differential responses of RB promoter constructs raise the possibility that the hGABP/E4TF1 site does not act as a positive regulatory element in JKT-1 cells, and the RB gene might be down-regulated at the transcriptional level in JKT-1 cells.