Purpose: Testing safety of Delta24-RGD (DNX-2401), an oncolytic adenovirus, locally delivered by convection enhanced delivery (CED) in tumor and surrounding brain of patients with recurrent glioblastoma. Patients and Methods: Dose-escalation phase I study with 3+3 cohorts, dosing 107to 1 × 1011viral particles (vp) in 20 patients. Besides clinical parameters, adverse events, and radiologic findings, blood, cerebrospinal fluid (CSF), brain interstitial fluid, and excreta were sampled over time and analyzed for presence of immune response, viral replication, distribution, and shedding. Results: Of 20 enrolled patients, 19 received the oncolytic adenovirus Delta24-RGD, which was found to be safe and feasible. Four patients demonstrated tumor response on MRI, one with complete regression and still alive after 8 years. Most serious adverse events were attributed to increased intracranial pressure caused by either an inflammatory reaction responding to steroid treatment or viral meningitis being transient and self-limiting. Often viral DNA concentrations in CSF increased over time, peaking after 2 to 4 weeks and remaining up to 3 months. Concomitantly Th1- and Th2- associated cytokine levels and numbers of CD3+T and natural killer cells increased. Posttreatment tumor specimens revealed increased numbers of macrophages and CD4+and CD8+T cells. No evidence of viral shedding in excreta was observed. Conclusions: CED of Delta24-RGD not only in the tumor but also in surrounding brain is safe, induces a local inflammatory reaction, and shows promising clinical responses.