Cytokines play a role in alopecia areata. We used immunohistochemical and in situ hybridization studies to demonstrate the persistence of pro-inflammatory as well as apoptotic mechanisms in skin biopsies from patients with chronic alopecia areata. In situ hybridization allows the visualization of the distribution of immunocompetent cells in vivo. We studied skin biopsies from 11 untreated alopecia areata patients and two normal controls. In situ hybridization was performed on frozen sections using 35S-radio-labeled riboprobes, specific for IL-1beta, IL-2, IL-6, INFgamma, and granzyme B mRNA. Immunohistochemistry was carried out using an anti-IL-1beta monoclonal antibody, and a monoclonal antibody directed against the human Fas protein. We demonstrated the presence of cells labeled with IL-1beta, IL-6, INFgamma, and granzyme B antisense probes. Similarly, cells labeled with anti-IL-1beta were found in 10 of 11 cases. The labeled cells were located in the mononuclear peri- and intrafollicular infiltrate. Cells expressing granzyme B were found in close contact with the follicle. Fas positivity was demonstrated in four of four cases at the level of the cytoplasmic membrane of the hair follicle keratinocytes. These results, based on visualizing the labeled cells, demonstrate that pro-inflammatory cytokines are produced by the mononuclear cell infiltrate in close contact with follicles in alopecia areata. Furthermore, they demonstrate for the first time that apoptotic mechanisms involving granzyme B and Fas-Fas ligand pathways may play a major role in the persistence of chronic alopecia areata.