Optical coherence tomography (OCT) is an emerging technology for high-resolution, noncontact imaging of transparent and scattering media. Polarization-sensitive optical coherence tomography (PS-OCT) is a functional extension of OCT that can image birefringent properties of a biological sample. PS-OCT was used to measure and image retardation and birefringent axis orientation of in vitro human cornea. We used a two-channel PS-OCT system employing a phase-sensitive recording of the interferometric signals in two orthogonal polarization channels. Using an algorithm based on a Hilbert transform, it is possible to calculate the retardation and the slow axis orientation of the sample with only a single A-scan per transversal measurement location. While the retardation information is encoded in the amplitude ratio of the two interferometric signals, the axis orientation is encoded entirely in their phase difference. We present maps of retardation and the distribution of slow axis orientation of the human cornea in longitudinal cross-sections and en face images obtained at the back surface of the cornea. The retardation increases in a radial direction and with depth; the slow axis varies in the transversal direction. Knowledge of the retardation and the slow axis distribution of the cornea might improve nerve fiber polarimetry for glaucoma diagnostics and could be useful for diagnosing different types of pathologies of the cornea. © 2004 Society of Photo-Optical Instrumentation Engineers.