New techniques for reconstructing large defects of the floor of the mouth include the use of cultured mucosal substitutes. The purpose of this study was to compare dispase and thermolysin for keratinocyte isolation. Keratinocyte yield per surface area of rabbit buccal mucosa was assessed by histology, cytokeratin 13 (CK13) staining, seeding efficiency analysis and cell diameter quantification. Surface areas of cultured mucosa were calculated. Histology showed that treatment by thermolysin resulted in incomplete separation of epidermis from dermis. Also, the absolute number of keratinocytes/cm(2) isolated mucosa, cell yield, cell size and seeding efficiencies was higher in the dispase group. A 3.45-fold larger graft could be reconstituted using dispase. The use of dispase, rather than thermolysin, to isolate cells from buccal mucosa is concluded to be favourable.