Vitiligo is an acquired idiopathic hypomelanotic disorder characterized by circumscribed depigmented macules resulting from the loss of cutaneous melanocytes. Although the exact cause of vitiligo remains obscure, autoimmunity may play a role in the development of the disease. The present study was undertaken to investigate the applicability of phage display technology to identify B-cell autoantigens in vitiligo. A melanocyte cDNA phage display library was subjected to rounds of enrichment with vitiligo patient IgG. Subsequently, enriched IgG-binding peptides representing putative autoantigens were identified by sequencing their encoding cDNAs. Radioimmunoassays were used to confirm the immunoreactivity of vitiligo patient (n=61) and control (n=28) sera to several of the putative autoantigens. Non-segmental vitiligo patient sera (n=53) were positive for antibody (Ab) reactivity to gamma-enolase (8%); alpha-enolase (9%); heat-shock protein 90 (13%); osteopontin (4%); ubiquitin-conjugating enzyme (15%); translation-initiation factor 2 (6%); and GTP-binding protein, Rab38 (15%). Ab reactivity to at least one of the previously unknown autoantigens was detected in 51% of patients with non-segmental vitiligo. In contrast, Ab reactivity in a group of patients with segmental vitiligo (n=8) was not demonstrated. Overall, the study indicated that the targets of autoantibodies in vitiligo patients can be revealed by employing the methodology of phage display.