The melanoma patient's immune response to tumor has been extensively studied. Yet, the frequently observed coexistence of tumor-associated Ag (TAA)-specific T cells with their target cells in vivo remains unexplained. Loss of TAA expression might contribute to this paradox. We studied TAA expression in metastases by obtaining fine-needle aspirations from 52 tumor lesions in 30 patients with melanoma before and soon after immunotherapy. Limitations due to low amounts of starting material were overcome with a high fidelity antisense RNA amplification method. TAA expression was measured by quantitative real-time PCR of anti-sense RNA. Decrease in gp100/Pmel-17 TAA preceded tumor disappearance in several instances and could be best explained by immune selection because most patients had received gp100/Pmel-17-specific vaccination. Conversely, immune selection was absent in nonregressing lesions. These observations suggest that vaccination, when successful, triggers a broad inflammatory reaction that can lead to tumor destruction despite immune selection. Additionally, lack of clinical response might be attributed to lack of this initiating event rather than immune escape. This study provides an insight into the natural history of tumors and defines a strategy for the characterization of gene expression in tumors during therapy.