TIMPs and PTEN are known to be inhibitors of the invasive activities of malignant glioma. But there has been no literature reported concerning the effect of combined gene transfer of these two genes on invasiveness of glioma. This study was designed to evaluate the effect of adenovirus-mediated in vitro gene transfer of tissue inhibitor of metalloproteinases-2 (TIMP-2) and phosphatase and tensin homology deleted on chromosome ten (PTEN) on invasion of human U87 glioma cells. The mRNA and protein expressions of TIMP-2 and PTEN in U87 cells infected with AdTIMP-2 and AdPTEN were determined by RT-PCR and Western blot, respectively. The relative activity of MMP-2 and MMP-9 were measured by Gelatin zymogram and invasion of U87 in vitro were detected using Boyden chamber. The number of invasion cell of U87, U87 infected with Ad-gal, AdPTEN, AdTIMP-2 and AdPTEN/TIMP-2 was 55.63+/-13.27, 48.27+/-14.75, 35.27+/-10.94, 27.37+/-12.81, and 19.17+/-5.45, respectively. In vitro invasiveness of glioma cells was significantly inhibited by infection with AdTIMP-2 and/or AdPTEN, which was not consistent with the change of MMPs activity. And in the combinated group, the inhibition effect was more remarkable than in single group. Our studies suggest that adenovirus-mediated combined TIMP-2 and PTEN gene therapy is possibly useful for anti-invasion therapy of malignant glioma.