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.