Recent reports have shown that natural killer (NK) cells may be long-lived, possess memory-like features and may need microbial priming to become fully reactive. Thus, the notion that these cells are typically innate, nonadaptive lymphocytes has been challenged. If microbial priming is essential for functional maturity, it is necessary to raise the question whether NK cells of laboratory mice, kept under strict hygienic conditions, represent these cells adequately. In their natural habitat, mice will encounter microbes to a greater extent, and we here investigated whether NK cells of feral mice showed signs of being primed. In comparison with C57BL/6 mice raised under specific pathogen-free conditions, NK cells from feral mice had high expression of CD69, KLRG1, granzyme B and NKp46 and a higher proportion of CD27+ cells, mostly CD11b−, as well as a higher presence in peripheral lymph nodes. Following cytokine stimulation, feral mouse NK cells had quickly inducible CD25 expression and a stronger interferon-gamma response. These findings indicate a high degree of pre-activation of NK cells of free-living mice, indicating a strong environmental impact on NK cells, which may be highly relevant for interpretation of studies in the mouse model.