BACKGROUND: Eliciting doses (ED) of allergenic foods can be defined by the distribution of threshold doses for individuals within a specific population. ED05 is the dose that elicits a reaction in 5% of allergic subjects. The predicted ED05 for peanut (PN) is 1.5 mg of peanut protein (6 mg whole peanut). OBJECTIVE: We sought to validate the predicted peanut ED05 (1.5 mg) with a novel single dose challenge. METHODS: Consecutive eligible peanut allergic children in 3 centres were prospectively invited to participate, irrespective of previous reaction severity. Predetermined criteria for objective reactions were used to identify ED05 single dose reactors. RESULTS: 518 children (mean age 6.8 years) were eligible. No significant demographic or clinical differences were identified between 381(74%) participants and 137 (26%) non-participants or between subjects recruited at each centre. 378 children (206 male) completed the study. Almost half the group reported ignoring precautionary allergen labelling. 245 (65%) experienced no reaction to the single dose of peanut. 67 (18%) reported a subjective reaction without objective findings. 58 (15%) experienced signs of a mild and transient nature that did not meet the pre-determined criteria. Only 8 subjects (2.1%, 95% CI 0.6%-3.4%) met the pre-determined criteria for an objective and likely related event. No child experienced more than a mild reaction, 4 of the 8 received oral antihistamines only and none received epinephrine. Food allergy related quality of life improved from baseline to 1 month post challenge regardless of outcome (eta squared = 0.2, p <0.0001). Peanut SPT, peanut and Ara h 2 spIgE levels were not associated with objective reactivity to PN ED05. CONCLUSION: A single administration of 1.5 mg PN protein elicited objective reactions in fewer than the predicted 5% of peanut-allergic subjects. The novel single dose OFC appears clinically safe and patient-acceptable, regardless of the outcome. It identifies the most highly dose-sensitive food allergic population, not otherwise identifiable using routinely available peanut SPT or spIgE levels but this single-dose approach has not yet been validated for risk assessment of individual patients.