The utility of immunostaining for S-100 protein for the identification of Langerhans' cells (Lcs) is limited by the distribution of this protein in other cell types. A new antibody (O10), directed against the more selectively expressed CD1a antigen, has become available for use with paraffin sections. Immunoperoxidase staining for S-100 protein and O10 antibody was performed on paraffin sections of lung specimens diagnosed as Langerhans' cell histiocytosis (LCH) (N-13), respiratory (smokers') bronchiolitis (n=5), and nonsmoking adult control subjects (n-2). Ten of the LCH patients were adults with primary pulmonary LCH (PLCH) and three were infants with disseminated LCH. Representative sections were also evaluated by confocal microscopy after dual labeling (for S-100 protein and O10 antibody) by immunofluorescence techniques. In each disorder, accumulations of O10- and S-100 protein-positive LCs were demonstrated in the interstitium and air spaces. The proportion of positive-staining cells in the bronchiolar mucosa with each antibody was significantly increased in biopsies from patiens with RB compared to those of non-smoking controls (p less than 0.001). This suggests that cigarette smoking is associated with hyperplasia of LCs in the bronchiolar mucosa. In addition, the proportion of S-100 protein-positive cells in the bronchiolar mucosa from normal controls was significantly increased compared to that of O10 antibody-positive cells (P less than 0.001). Our impression that many of these cells are dendritic cells is supported by the finding of S100 protein-positive, O10 antibody-negative cells with prominent dendritic morphology, as demonstrated in RB by confocal microscopy and double labeling. Confocal microscopy showed that the LCs were more evenly and homogeneously stained for O10 antibody than for S-100 protein and were easily distinguished from S-100 protein-positive, O10 antibody-negative macrophages and dendritic cells. Immunoreactivity of the O10 antibody is at least as sensitive and more specific than that for S-100 protein in the evaluation of disorders of pulmonary LCs.