Adoptive cell therapy with tumor-infiltrating lymphocytes (TIL) can mediate objective responses in up to 50% of malignant melanoma patients with a good performance status refractory to standard treatments. Current protocols for generation of TILs rely on open surgery for access to tumor tissue. We obtained tumor material by ultrasound-guided core needle biopsy or surgery from melanoma patients with progressive disease and were able to isolate >5 × 10(6) TILs from 23 of 24 patients who were subsequently treated with these cells. One-third of the individual TIL-positive cultures displayed interferon gamma activity after stimulation with relevant melanoma cell lines. When expanded TILs were used for treatment in combination with daily low dose s.c. IL-2 after prior lymphodepleting chemotherapy, we observed objective clinical responses in one patient treated with TILs obtained from surgery and 4 patients treated with TILs from core biopsies. The results of this study demonstrate for the first time the potential of core biopsies for generation of relevant numbers of TILs that can mediate objective responses in patients with metastatic malignant melanoma. Ultrasound-guided core needle biopsy is a robust, safe and inexpensive approach to obtain tumor tissue for TIL generation, and is especially valuable in instances where surgery is contraindicated.