Recent studies have revealed that glucose transporter 1 (GLUT 1) expression can be detected in basal layer cells of skin. Expression levels of GLUT 1 are thought to be regulated by glucose transport activity within cells. Glucose metabolism in the epithelial layers may affect keratinocyte differentiation. We studied the localization of GLUT 1 expression in the epithelial layers of oral mucosa equivalents, grown in a defined serum-free culture medium without a feeder layer. The oral mucosa produced in this system is minimally exposed to potential foreign contaminants and is thus more acceptable for use in humans. Samples of native human gingiva and oral mucosa equivalents on days 4, 11, and 18 were fixed in 4% paraformaldehyde, embedded in paraffin, and cut into 5-micrometer-thick sections. For immunohistochemical studies, sections were stained by the ABC method using polyclonal antibodies to rabbit GLUT-1. Well-stratified, differentiated epithelial layers in day 18 oral mucosa equivalents had a similar keratinization pattern to that of native tissue. GLUT 1 was expressed in both the basal cell layer and suprabasal cell layer of oral mucosa epithelium, but not in the superficial layer. Immunoreactivity for GLUT-1 was detected in all epithelial layers of oral mucosa equivalents, except for the superficial layer of day 18 equivalents. In conclusion, keratinocytes of all layers of oral mucosa equivalents were in a highly active metabolic state and showed increased glycolysis, suggesting active proliferative and a less differentiated state as comnared with the basal and na.ra.ba.sa.1 cell lavers of native tissue.