Vitiligo is an acquired dermatological disorder characterized by a loss of epidermal melanocytes resulting in depigmentation of the skin. Mechanisms underlying the destruction of melanocytes in vitiligo remain unclear. An animal model to study spontaneously occurring autoimmune vitiligo is the mutant Smyth line (SL) of chickens. This investigation was designed to determine whether the pathogenesis of depigmentation in Smyth line chicken vitiligo (SLV) involves an apoptotic mechanism. Terminal deoxynucleotide transferase-mediated fluorescein-dUTP nick end labeling (TUNEL) was used to detect in situ cell apoptosis in cryostat sections of 2-week-old regenerating feathers. Two-week-old regenerating feathers were obtained from SL chickens and their normally pigmented controls including the parental Brown line (BL) and Light Brown Leghorn (LBL) chickens at 6, 8, 10 and 12 weeks of age. In feathers from vitiliginous SL chickens, the number of TUNEL+ cells was significantly (P<or=0.05) higher than that in the feathers of non-vitiliginous SL, BL or LBL chickens. These TUNEL+ cells were primarily located in the epithelial barb ridge where melanocyte cell bodies are located. The extent of this apoptosis in the feathers of SLV chickens varied with the severity of depigmentation of the feathers (i.e., highest in active depigmentation), suggesting a close association between apoptosis and the disappearance of melanocytes. In addition to TUNEL staining, most sections were double-stained with monoclonal antibodies specific to either CD8 or MHC class II molecules to further explore the relationship between CD8+ feather-infiltrating lymphocytes and this increase in apoptotic cells. Compared to normally pigmented controls, the number of CD8+ and MHC class II+ cells in the feather pulp and the barb ridge increased 2-4 weeks before the visible onset of SLV, and was directly related to the changes in the number of TUNEL+ cells prior to, at onset and during depigmentation. Moreover, some of these infiltrating CD8+ cells were localized next to or near the TUNEL+ cells. These observations suggest that enhanced apoptosis in the feather of SLV chickens is a pathogenic mechanism involved in the death of melanocytes and appears to be induced by infiltrating cytotoxic T lymphocytes (CD8+).