Congenital perilymphatic fistula (PLF) as a diagnosis for progressive, fluctuating, or sudden sensorineural hearing loss with or without vertigo is still controversial. Beta-2 transferrin is a protein that is unique to cerebrospinal fluid, aqueous humor, and perilymph. A recent pilot study demonstrated that beta-2 transferrin may be an objective test to determine the existence of a congenital PLF. The authors prospectively evaluated and recommended surgery for 43 children with suspected PLF over the past 3 years. A prospective, blinded study was performed by having the attending otolaryngologist evaluate the middle ear at the time of surgery for a PLF and any middle ear abnormalities. Samples of fluid were collected from the oval and round windows and were tested for beta-2 transferrin. Of 16 patients undergoing tympanoplasty or tympanomastoidectomy who served as controls, none were positive for beta-2 transferrin. Of the 43 patients undergoing exploratory tympanoplasty for PLF, 20 (46.5%) were considered to be negative for PLF on microscopic visualization; 23 (53.5%) were found to be positive. Of the 20 patients thought to be negative for PLF, 18 (90%) tested negative for beta-2, but 2 of these patients were positive for beta-2, and both had a congenital middle ear abnormality. Of the 23 patients found to have a PLF at surgery, 6 (26.1%) tested positive for beta-2, and all of these 6 had middle ear abnormalities. Of the 17 patients negative for beta-2, 9 had normal anatomy; 6 had middle ear abnormalities, and 2 had erosive changes. The authors conclude that beta-2 transferrin, an objective test, confirms the existence of congenital PLF in children, which is associated with middle ear abnormalities.