The aim of this study was to determine any differences in the collagen molecular and fibrillar packing, or the arrangement of the proteoglycans along the fibril axis, in the stroma of keratoconus and control corneas. High and low-angle x-ray diffraction patterns from the fibrillar and molecular packing of collagen in keratoconus and control corneas were obtained using a synchrotron radiation source. The results indicate no difference in interfibrillar spacing between keratoconus and control corneas at normal physiological hydration, or over a range of hydrations (H = 1-11). This unambiguously demonstrates that the thinning of the stroma that occurs in keratoconus is not a result of closer packing of the collagen fibrils in the stroma. Intermolecular spacings were shown to be significantly (P < 0.001) lower in keratoconus corneas at normal physiological hydration and over a range of hydrations (H = 1-11). Meridional patterns from the axial distribution of electron density along the collagen fibrils were obtained from untreated control and keratoconus corneas and from the corneas after their proteoglycans were stained with cupromeronic blue. Analysis of the integrated intensities of the first nine orders of these reflections show there is a difference in the staining behavior of collagen-associated proteoglycans in control and keratoconus corneas. Determination of the electron density vectors along the collagen fibrils of cupromeronic blue-stained corneas by the use of Patterson functions indicates that the keratoconus corneal stroma has a specific, ordered proteoglycan that is present in lower numbers along the collagen fibrils, and that it stains less with cupromeronic blue or is in a more disordered arrangement than in the controls.