In a prospective phase I/II clinical study, we treated eight patients suffering from recurrent glioblastoma multiform with stereotactically guided intratumoral convection‐enhanced delivery of an HSV‐1‐tk gene–bearing liposomal vector and systemic ganciclovir. Noninvasive identification of target tissue together with assessment of vector‐distribution volume and the effects of gene therapy were achieved using magnetic resonance imaging and positron emission tomography. The treatment was tolerated well without major side effects. In two of eight patients, we observed a greater than 50% reduction of tumor volume and in six of eight patients focal treatment effects. Intracerebral infusion of contrast medium before vector application displayed substantial inhomogeneity of tissue staining indicating the need of test infusions to monitor the mechanical distribution of vectors. Visualization of therapeutic effects on tumor metabolism and documentation of gene expression using positron emission tomography indicated that molecular imaging technology appears to be essential for the further development of biological treatment strategies.