Purpose. Interferometric methods have considerable potential for studying the thickness of layers of the human tear film and cornea because of their ability to make noninvasive, accurate, and rapid measurements. However, previous interferometric studies by Prydal and Danjo yielded tear thickness values near 40 and 11 μm, respectively, considerably greater than estimates made by invasive methods of 4 to 8 μm. Using a modified version of Danjo's method, interference effects from the tear film and cornea were studied, with the aim of correlation with known structure and optical properties of the cornea and hence determining the most probable value of tear film thickness. Methods. Reflectance spectra from the human cornea were measured at normal incidence. These spectra show oscillations whose maxima correspond to constructive interference between light reflected from the air surface and from some deeper surface. The frequency of these spectral oscillations is proportional to the thickness of the layer between the air surface and the second surface. Therefore, Fourier analysis of reflectance spectra can be used to determine the thickness of layers of the tear film and cornea. In the main experiment, 36 low-resolution spectra were obtained from six normal eyes for measuring thickness up to 100 μm. Control experiments included measurements of the time course of thickness changes and high-resolution spectra for measuring thickness up to 1000 μm. Results. For the main experiment, in the thickness range 1 to 100 μm, the strongest peak in the Fourier transform was near 3 μm (range, 1.5-4.7 μm) beneath the air surface. In the range 20 to 100 μm, the strongest peak was near 55 μm (range, 50-59 μm) for all 36 spectra; none were in Prydal's range near 40 μm. This 55-μm peak is consistent with a reflection from the basement membrane of the epithelium. Time course measurements after a blink show that the 3-μm peak is not an artifact. High-resolution spectra gave a peak near 510 μm, corresponding to the complete thickness of the cornea (plus tear film). This peak had a contrast similar to that of the 3-μm peak. Conclusions. These studies did not confirm Prydal's estimate of approximately 40 μm. Nor were there prominent peaks near Danjo's value of approximately 11 μm, except in cases of probable reflex tears. Because the reflection at the aqueous-mucus boundary would be expected to be weaker than that from the epithelial surface, the 3-μm peak is unlikely to correspond to the aqueous layer (rather than the complete tear film). The proposal that the 3-μm peak corresponds to a reflection from the front of the cornea is supported by the demonstration of a peak of similar contrast from the back of the cornea. Thus, the current evidence consistently supports a value of approximately 3 μm for the thickness of the human precorneal tear film.