In order to investigate the effects of in vivo treatment with interferon-alpha (IFN-alpha) on melanoma antigens, a clinical EORTC trial (No. 18852) was accompanied by an immunohistological study. Twenty patients with melanoma metastases of skin and soft tissues, eventually also of the lung, who were treated with systemic IFN-alpha, were evaluated for a comparison of metastases before (40) and during (42) treatment. Representative cryostat sections were studied immunohistologically with a panel of monoclonal antibodies against differentiation antigens (HMW-MAA, K-1-2, NKI-beteb, M-2-10-15), progression markers (transferrin receptor, ICAM-1, VLA-2), histocompatibility antigens (HLA-A, B, C, HLA-DR) and the proliferation-associated nuclear antigen Ki67. We found an overall reduction of the proliferation-associated antigen Ki-67 (p < 0.01), and an increase in expression of HLA-DR (p < 0.05) and ICAM-1 (trend) during treatment. The intensity of expression of HLA-A, B and C antigens as well as pigmentation (p < 0.01) was found to be increased. Early progression (< or = 8 weeks after onset of treatment) was associated with a lack of phenotypic changes. The data suggest an independent modulation of proliferation, pigmentation, and antigen expression by systemic treatment of metastatic melanoma with IFN-alpha.