A diagnosis of perilymphatic fistula is still controversial. Recently, a case report indicated that β-trace protein (prostaglandin D synthase) might be a potential marker for perilymphatic fluid. In this multicentre clinical case series study β-trace protein was used as a marker for perilymphatic fluid fistula. Fifteen fluid samples were collected during diagnostic tympanoscopy. In addition, five samples were collected from patients with tympanic membrane perforation for use as as negative controls. Samples were obtained using precision glass capillaries and were analysed for β-trace protein using laser nephelometry. The diagnosis of perilymphatic fistula was defined by the patient's history, the audiological and vestibular investigation and the findings at tympanoscopy. The cut-off level of β-trace protein for perilymph-positive samples was chosen at 1.11 mg/l. The sensitivity and specificity were calculated using a 2 × 2 contingency table. There was no false positive result, but in two cases a false negative result was found. The specificity was 1 and the sensitivity was 0.81. The material of this first clinical study is small owing to the rarity of patients undergoing diagnostic tympanoscopy for perilymphatic fluid fistula. However, according to these preliminary results β-trace protein might be a promising marker in the diagnosis of perilymphatic fluid fistulas.