Flap endonuclease 1 (FEN1) is a DNA replication/repair protein involved in Okazaki fragment processing, long-patch base excision repair, DNA double-strand break repair and stalled replication fork restart. FEN1 is also important for preservation of telomere stability and thus a key node in maintaining genomic stability. The aim of the present study was to elucidate the level of expression of FEN1 protein in cancer of testis, lung and brain. FEN1 protein expression was studied by Western blot analysis in specimens of tumor tissues compared with the normal tissue from the same patient or normal brain extract. In addition, FEN1 was transiently down-regulated in the glioblastoma cell line LN308 by transfection with siRNA. The transfected cells were treated with cisplatin, temozolomide, nimustine and methyl methanesulfonate (MMS). Induced apoptosis (subG1 fraction) was analysed by flow cytometry. Our data show a clear overexpression of FEN1 in 19/25 samples from testicular tumors (mostly seminomas) and 4/4 samples from lung tumors (non-small cell lung cancer). For brain tumors, 9/11 glioblastoma multiforme and 5/8 astrocytomas expressed FEN1 protein at a higher level than did normal brain tissue. Overall, the data demonstrate that FEN1 overexpression is common in testis, lung and brain tumors. Low-level expression of FEN1 by siRNA down-regulation increased sensitivity to methylating agents (temozolomide, MMS) and cisplatin in LN308 glioma cells, which indicates that altered FEN1 expression might impact the therapeutic response.