Natural killer (NK) cell infusions can induce remissions in subsets of patients with different types of cancer. The optimal strategies for NK cell activation prior to infusion are still under debate. There is recent evidence that NK cells can acquire long-term functional competence by preactivation with the cytokines IL-12/15/18. The mechanisms supporting the maintenance of long-term NK cell antitumor activity are incompletely under-stood. Here, we show that NK cells preactivated with IL-12/15/18, but not with IL-15 alone, maintained high antitumor activity even 1 mo after transfer into lymphopenic RAG-2γc mice. The NK cell intrinsic ability for IFNγ production coincided with demethylation of the conserved non-coding sequence (CNS) 1 in the locus, previously shown to enhance transcription of . In a xenograft melanoma mouse model, human IL-12/15/18-preactivated NK cells rejected tumors more efficiently. In RAG-2γc mice, co-transfer of CD4 T cells further improved the long-term competence of NK cells for IFNγ production that was dependent on IL-2. CD4 T cell activation during homeostatic proliferation required macrophages and further promoted the long-term NK cell antitumor activity. Thus, NK cells can "remember" a previous exposure to cytokines by epigenetic imprinting resulting in a remarkable stability of the IFNγ-producing phenotype after adoptive transfer. In addition, our results support combination of cytokine-preactivated NK cells with CD4 T cell activation upon lymphopenic conditioning to achieve long-term NK cell effector function for cancer immunotherapy.