Abstract Purpose: The 14 amino acid sequence (aa450–463) TKDNNLLGRFELSG (TKD) of heat shock protein 70 (Hsp70) was identified as a tumor-selective recognition structure for natural killer (NK) cells. Incubation of peripheral blood lymphocyte cells with TKD plus low-dose interleukin 2 (IL-2) enhances the cytolytic activity of NK cells against Hsp70 membrane-positive tumors, in vitro and in vivo. These data encouraged us to test tolerability, feasibility, and safety of TKD-activated NK cells in a clinical Phase I trial. Experimental Design: Patients with metastatic colorectal cancer (n = 11) and non-small cell lung cancer (n = 1) who had failed standard therapies were enrolled. After ex vivo stimulation of autologous peripheral blood lymphocytes with Hsp70-peptide TKD (2 μg/ml) plus low-dose IL-2 (100 units/ml), TKD was removed by extensive washing, and activated cells were reinfused i.v. The procedure was repeated for up to six cycles, applying a dose escalation schedule in 4 patients. Results: The percentage of activated NK cells in the reinfused leukapheresis products ranged between 8 and 20% of total lymphocytes, corresponding to total NK cell counts of 0.1 up to 1.5 × 109. Apart from restless feeling in 1 patient and itching in 2 patients, no negative side effects were observed. Concomitant with an enhanced CD94 cell surface density, the cytolytic activity of NK cells against Hsp70 membrane-positive colon carcinoma cells was enhanced after TKD/IL-2 stimulation in 10 of 12 patients. Concerning tumor response, 1 patient was in stable disease during therapy by formal staging criteria and another patient showed stable disease in one metastases and progression in another. Conclusions: Reinfusion of Hsp70-activated autologous NK cells is safe. Immunological results warrant additional studies in patients with lower tumor burden.