For glioma- and glioblastoma-specific gene expression, we utilized a nestin regulatory element whose activity was evaluated by the reporter gene lacZ. Nestin is a 38-kDa intermediate filament protein, and is expressed specifically in the neuroepithelial stem cells. Nestin is detected in gliomas and glioblastomas, but not in normal brain tissue. We constructed a nestin gene regulator by placing nestin's second intron before the 5' upstream region (2iNP). To obtain enhanced expression of this tissue-specific regulator, we utilized the adenovirus double-infection method with a Cre-loxP on/off switching system. We constructed a 'regulator' vector, Ax2iNPNCre, which expresses Cre recombinase under the control of the nestin regulatory element, 2iNP. A 'reporter' vector, AxCALNLNZK, expresses lacZ under the control of a strong CAG promoter when the stuffer sequence has been removed by Cre recombinase at a pair of loxP sites. We used seven human glioma/glioblastoma cell lines: U251, KG-1C, NGM5, U87 MG, LN-Z308, NP-2 and T98G. Of these, nestin was expressed highly in U251 and KG-1C, less in NGM5, and undetectably in the other four lines. With the use of the two adenovirus vectors, we found X-gal staining and high nestin regulator-promoted beta-galactosidase activities in four of the seven glioma/glioblastoma cell lines. Staining was strong in U251, KG-1C and NGM5, and less in U87 MG. LacZ expression was nearly undetectable in the non-glioma cell line, HeLa, but a little in COS-7. The adenovirus double-infection method, which uses a nestin regulator, is applicable for glioma/glioblastoma-specific expression.