Localized absence of epithelial Langerhans cells (LC) has been shown to affect systemic immune responses, allow microbial colonization and play a possible role in carcinogenesis. Because use of smokeless tobacco is associated with abnormal oral mucosal changes and development of carcinoma, we examined lesion and control specimens from 17 current users of smokeless tobacco to determine whether lesions showed changes in LC number or antigen expression. We identified LC by immunohistochemistry with antibodies to the antigens T6, HLA-DR, HLA-DQ, and HLA-DP. Lesion specimens contained fewer LC (means of 6 LC/mm and 10 LC/mm2) than did the corresponding control specimens (means of 14 LC/mm and 30 LC/mm2), and in each pair of lesion and autologous control specimens the reduction in LC was on average 58% (range, 3% to 95%). There were no apparent differences between lesion and control specimens in the number of LC expressing each of the four marker antigens. Reductions in LC occurred in all types of smokeless tobacco-associated lesions, regardless of increased epithelial thickness or changes in keratinization. Our data indicate that smokeless tobacco reduces the number of Langerhans cells at its site of contact with the oral mucosa.