A 40-year-old man developed a Horner syndrome as part of a dorsolateral medullary brainstem infarction. Thirty-six hours after the onset of the stroke, topical instillation of 0.5% apraclonidine produced reversal of anisocoria. This is the first case in which apraclonidine testing has been applied to a patient with a Horner syndrome caused by a lesion in the first segment of the oculosympathetic pathway and the shortest reported interval between clinical manifestations of the lesion and apraclonidine-induced reversal of anisocoria. A review of all reported cases of apraclonidine testing in Horner syndrome suggests that this is a promising diagnostic adjunct that must be validated in larger studies.