Melanoma is a highly malignant disease that may initially present as a poorly differentiated metastatic tumor. Therefore, the S100 immunostain, immunoreactive in 96% to 99% of melanoma, is used to evaluate poorly differentiated malignant tumors. To develop criteria for correctly diagnosing S100-negative melanomas, we studied the immunohistochemical profile of 1553 patients enrolled in ongoing National Cancer Institute clinical trials for melanoma. Seventeen patients (1%) had metastatic melanoma specimens that were negative for S100. Of the 17 S100-negative lesions, 10 (59%) were immunoreactive for both GP100 and MART-1. Of the 17 S100-negative cases, 13 had a documented primary melanoma. Twenty-four percent of the S100-negative cases had an ocular primary, whereas only 6% of all melanomas had an ocular origin. In 11 of the 17 cases with previous surgical specimens, a prior documented S100-immunoreactive specimen was identified in 9 cases (82%). The time interval for loss of S100 immunoreactivity ranged from 3 weeks to 3 years (average, 13.5 months). There was no association between S100-negative status and histological appearance or site of metastasis. We conclude that all S100-negative melanomas could be correctly identified by negative workup for carcinoma, lymphoma, and sarcoma plus (1) GP100/MART-1 immunoreactivity and/or (2) prior documentation of melanoma.