X-chromosome-inactivation assays can be used to assess clonality. We used such an assay at the human androgen-receptor gene locus in three female patients with histologically proven Langerhans cell histiocytosis. All patients were heterozygous for this locus. Cells bearing the Langerhans cell phenotype were purified from involved tissue after fluorescence-activated cell sorting with monoclonal antibodies against the CD1a complex. After HhaI digestion of DNA, these CD1a positive cells demonstrated a non-random X-chromosome-inactivation pattern, whereas CD1a negative cells in the same tissue showed a random pattern. Our data suggest that Langerhans cell histiocytosis represents a clonal proliferation of cells bearing the Langerhans cell phenotype.