Expression of the Wilms' tumor suppressor WT1 has been demonstrated in a variety of tumors and tumor cell lines, e.g., in breast cancer and melanoma cell lines. Its role is controversial, with evidence for both tumor-promoting and tumor-suppressing activities. In this paper, we show that WT1 is expressed in malignant melanoma in >80% of the tumor cells, but not in normal skin or benign melanocytic nevi in vivo. We detected an unusual shift of WT1 isoforms towards WT1(+17AA/+KTS) in melanoma. WT1 shared an overlapping expression with proliferating nuclear cell antigen and with Nestin and Zyxin, which are involved in melanoma cell proliferation. To investigate whether WT1 is directly involved in melanoma cell proliferation, we made use of an RNAi approach in vitro. WT1 silencing significantly reduced the expression of Nestin and Zyxin and resulted in inhibition of melanoma cell proliferation as determined by a reduced BrdU incorporation. These findings suggest a direct role of WT1 in melanoma proliferation, which might be mediated via Nestin and Zyxin. Furthermore, expression of WT1 in vivo clearly discriminates between benign acquired nevi and malignant melanomas and appears to be correlated with melanocytic atypia and malignancy.