Inhibition of tumor neovascularization has profound effects on the growth of solid tumors. Our previous studies have shown the effect of VEGF165-PE38 recombinant immunotoxin on proliferation and apoptosis in human umbilical vein endothelial cells in vitro. In this study, we explored the direct inhibition of angiogenesis in chick chorioallantoic membrane and antiangiogenic therapy in a malignant glioma model. HEK293 cells were transfected with the pVEGF165PE38-IRES2-EGFP plasmid. ELISA was used to confirm the expression of VEGF165-PE38 in the transfected cells. These cells released 1396 +/- 131.9 pg VEGF165-PE38/1x10(4) cells/48 h into the culture medium and the supernatant was capable of inhibiting the growth of capillary-like structures in chick chorioallantoic membrane assay. In a murine malignant glioma model, plasmid was directly administered via multiple local intratumoral delivery. After day 16 the tumor volume in mice treated with pVEGF165PE38-IRES2-EGFP was significantly lower than that in mice in the control groups. Immunohistochemistry studies showed that the treated group had decreased expression of CD31. Quantitative analysis of microvessel density in the treated group was 1.99 +/- 0.69/0.74 mm(2), and was significantly lower than that in the control groups (9.33 +/- 1.99/0.74 mm(2), 8.09 +/- 1.39/0.74 mm(2) and 8.49 +/- 1.69/ 0.74 mm(2)). Immunohistochemistry analysis indicated that immunotoxin VEGF165-PE38 was distributed in the treated group in malignant glioma tissue. Our findings provide evidence that the in vivo production of VEGF165-PE38 through gene therapy using a eukaryotic expression plasmid had potential antiangiogenic activity in malignant glioma in vivo.