The most abundant soluble protein of bovine cornea, BCP 54 (Bovine Corneal Protein, molecular weight 54 kD) was isolated and digested under both limited and complete digestion conditions withStaphylococcus aureusV8 protease. The fragments resulting from limited digestion were separated by one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis, transferred to a polyvinylidene difluoride membrane, visualized by Coomassie Blue staining, cut out, and submitted to N-terminal protein sequence analysis. Complete digestion fragments were separated on a Vydac C18 reverse-phase HPLC, collected, and concentrated prior to sequencing. Using this method, we obtained amino acid sequence data from three internal V8 protease derived fragments of BCP 54 and a number of HPLC fragments. Comparison of these amino acid sequences, corresponding to 30% of the BCP 54 molecule, to those sequences contained within release 22 of the National Biomedical Research Foundation Protein Identification Resource revealed no extended sequence similarity of known proteins to BCP54.