A variety of human melanoma-associated antigens (MAA) have been identified that can be recognized by T lymphocytes in a major histocompatibility complex-restricted fashion. Among them, tyrosinase, MART-1/Melan- A, and gp100 are derived from nonmutated melanocyte lineage-specific antigens (Ag). These Ag can be recognized by CD8+ and, in the case of tyrosinase, CD4+ T cells. The in situ expression of these MAA may be a significant cofactor in determining the recognition of melanoma targets by Ag-specific T cells. In this study, we examined the patterns of expression of these MAA using immunohistochemical methods on 30 metastatic tumor deposits derived from 25 patients. MAA expression was heterogeneous among the 30 specimens and also within individual lesions. Of note, 23% of the samples examined failed to express the gp100 protein, and 17% of samples had no detectable expression of MART-1. In contrast, all lesions demonstrated some degree of tyrosinase expression even in cases where both gp100 and MART-1 were not detectable. In addition, 60% of samples (18 of 30) showed strong positivity for tyrosinase (> 75% of cells staining) compared with 40% for gp100 and 36% for MART-1. Currently, a number of experimental immunotherapies for melanoma are directed against the MAA tyrosinase, MART-1, and gp100. Although threshold levels of Ag required for T-cell recognition have not yet been defined, tumor-associated Ag expressed in high density, such as tyrosinase, may be better targets for future immunotherapy trials.