We quantitated the amount of collagen in corneas obtained from patients with keratoconus. Following a previously described procedure, we deparaffinized tissue sections and stained them with the collagen-specific dye Sirius red. Twenty-two (group I) of 31 keratoconus corneas examined were normal, whereas the remaining 9 (group II) showed reduced collagen staining per mm2of surface area compared with the controls. These results agreed with our previous data demonstrating heterogeneity in keratoconus in terms of collagen content analyzed biochemically in addition, we adapted another histochemical method, using safranin O, to measure the polyanion (mainly glycosaminoglycan) content in paraffin sections of the same specimens. All except two keratoconus corneas showed markedly higher polyanion staining per mm2of surface area than the controls. This suggests that keratoconus corneas may contain an increased amount of glycosaminoglycans and other polyanions. Such an abnormality may be related to some of the pathologic changes seen in these corneas.