Primary cultures of rabbit corneal endothelial cells were labeled with proline and the medium proteins were fractionated by sedimentation through sucrose density gradients. A new collagen, endothelial cell (EC) collagen, was completely separated from type IV collagen without denaturation by this technique. Sodium dodecyl sulfate electrophoresis demonstrated EC collagen fragments of 180K, 120K, and 60K daltons. Prior pepsin treatment produced only the 60K fragment after electrophoresis. The cyanogen bromide peptide pattern of the 180K EC peptide demonstrated that EC was different from type IV. A preliminary structural model for the largest helical form (∼ 540K daltons) of EC collagen is presented.