Mature cornea consists of orthogonally stacked lamellae formed by uniformly thin collagen fibrils evenly spaced in parallel. In tissue, collagens must be cross-linked to exhibit normal physical properties. In the extracellular matrix (ECM), cross-linking can result from the enzymatic activity of lysyl oxidase (LOX) and tissue transglutaminase (TG). The relationship between tissue-specific matrix organization and cross-links in the ECM of chicken embryonic cornea has been investigated. We found that typical sheets of orthogonally arranged collagen fibrils were formed by keratocyte cell cultures. Such lamellae were not apparent when aldehyde-derived cross-link formation was inhibited. However, the collagen fibrils formed were thicker with a clearly visible banding pattern with the TG inhibitor. Interestingly, the presence of both cross-link leads to striking effects on matrix formation visible by loss of matrix organization and an irregular fibril diameter distribution.