The cornified cell envelope (CE) is a specialized structure which contributes barrier function to stratified squamous epithelial cells. It is composed of an amalgam of several structural proteins that are rendered insoluble by isopeptide bond crosslinking by transglutaminases. One set of the structural proteins present in CEs of most such epithelia are the small proline rich (SPR) proteins, which are a family of about 12 related structural proteins. We have recovered a large number of peptides containing isopeptide crosslinks, including 236 involving SPR proteins, following proteolysis of CEs isolated from foreskin epidermal tissue and cultured epidermal keratinocytes. Analysis of this database has provided novel information on their function. First, we found that SPRs became crosslinked to many other structural proteins within the CE. Second, multiple glutamine and lysine residues located only on the amino- and carboxy-termini of the SPR proteins were involved in crosslinking, so that the two ends are functionally equivalent. Third, the SPRs functioned as cross-bridging proteins, by directly adjoining other CE structural proteins. In the specialized case of the epidermal CE, the SPRs cross-bridged between loricrin. In cultured keratinocytes which make little loricrin and serve as a model for internal stratified squamous epithelia, the SPRs formed extensive cross-bridges among themselves. Thus SPRs are ubiquitous cross-bridging proteins whose differential expression patterns apparently reflect specific barrier requirements of different epithelia.