In the present study, we describe the in vitro transcription–translation of human tyrosinase-related protein-1 (TRP-1) cDNA and subsequent use of the resulting 35S-labelled TRP-1 in a radioimmunoassay to analyse vitiligo sera for the presence of TRP-1 antibodies. Of 53 vitiligo sera examined in the assay, three (5.7%) were found to be positive for TRP-1 antibodies. In contrast, sera from 20 controls, 10 patients with Hashimoto's thyroiditis and 10 patients with Graves' disease were all negative for TRP-1 antibodies. Although glycosylation of the labelled protein was necessary for its immunoprecipitation by TRP-1-specific monoclonal antibody TA99, this post-translational processing did not affect the binding of any of the sera tested. All three patients positive for TRP-1 antibodies (aged 50–63 years) had had vitiligo of the symmetrical type for more than 1 year, and all of them also had an associated autoimmune disorder: Graves' disease in one and autoimmune hypothyroidism in two. In addition, antibodies to the melanogenic enzymes tyrosinase and tyrosinase-related protein-2 (TRP-2) were present in their serum. Absorption studies indicated that preincubation with COS-7 cell extract containing either expressed TRP-1, tyrosinase or TRP-2 absorbed out the immunoreactivity of the three sera positive in the radioimmunoassay (RIA) with [35S]TRP-1. The results indicate that autoantibodies to TRP-1 cross-react with tyrosinase and TRP-2, suggesting one or more common epitopes between the three proteins.