Measurement of the macular pigment optical density (MPOD) by heterochromatic flicker photometry (HFP) is accomplished by viewing a small circular stimulus that alternates between a test wavelength that is absorbed by the MP (typically--blue, 460 nm) and a reference wavelength that is not absorbed (typically-green, 540 nm). Flicker observed by the subject is reduced to a null point by adjusting the intensity of the former while viewing the stimulus centrally, and then peripherally. A higher intensity, I, of the blue component of the stimulus is needed under central viewing conditions owing to attenuation by the MP. The MPOD at the test wavelength is given by log (Icentral/Iperipheral). Variation of the test wavelength has been used to measure the MPOD spectrum. This in vitro MPOD spectrum matches that of the carotenoids present in the macular region of the retina and demonstrates the validity and specificity of this methodology. The distribution of MPOD in the retina can be determined with HFP using a series of annular stimuli of different diameters.