Background Neuroblastoma is the commonest cancer in infants. Survival in high-risk groups is low (40-50%). Newer treatments are needed to improve survival and morbidity. Cytomegalovirus (CMV) is a common viral infection, which increases gamma delta T cells. We investigated the use of CMV-reactive gamma delta T cells as a potential new immunotherapy. Methods Peripheral blood mononuclear cells from 30 paediatric haemato-oncology patients with or without CMV infection were analysed by flow cytometry. gamma delta T cells were expanded, and then co-cultured with CMV targets or neuroblastoma cells. T-cell activation, cytokine secretion, killing activity, and mechanisms were determined by ELISA, MTT colorimetric assay, and blocking assays. Novel gamma delta T cell receptors (TCR) were identified by sequencing. Findings In paediatric haemato-oncology patients, acute CMV led to higher proportions of total gamma delta T cells (p=0.0002) with the dominant subtype V delta 1 (p=0.0035). CMV-reactive gamma delta T cells were of the effector memory phenotype and expanded to clinically significant numbers for adoptive transfer. When compared with gamma delta T cells from CMV-negative patients, CMV-reactive gamma delta T cells had statistically significantly higher interferon gamma release (p<0.001) in co-cultures with CMV-infected fibroblasts and showed cytolytic activity against CMV-infected fibroblasts and neuroblastoma cells which was mediated by gamma delta TCR and the NKG2D receptor. Sequencing showed that the dominant gamma delta T-cell chains in CMV-infected patients were V delta 1 V gamma 2. Within the TCRs, CDR3 sequences had minimal diversity, gamma chains had wide variations, and we identified a novel subpopulation in some patients. Interpretation We show that acute CMV infection in paediatric haemato-oncology patients leads to an increase in the V delta 1 V gamma 2 subtype of gamma delta T cells. They can be expanded for adoptive transfer and are likely to retain killing ability post expansion. They recognise and kill CMV-infected targets and neuroblastoma cells via the gamma delta TCR and the NKG2D receptor. We have identified a novel TCR in a subpopulation of CMV-positive patients. Funding Great Ormond Street Charity, National Institute for Health Research Biomedical Research Centre, Olivia Hodson Cancer Fund.