The in vitro cultivation of murine spleen cells with MnCl2resulted in the enhancement of natural killer (NK) cell activity as measured in a 4‐h51Cr‐release assay. Optimal enhancement of NK activity was observed at concentrations of 70–20 μg MnCl2/culture (72–144 μM Mn2+). Enhancement of NK activity by MnCl2was not associated with any changes in the number or viability of cells following culture. The addition of antiasialo GM1, antibody and complement to spleen cell cultures completely abrogated the enhancement of NK activity by MnCl2. The enhancement of NK activity by MnCl2in vitro was accompanied by interferon induction. The addition of rabbit anti‐mouse interferon to spleen cells cultured with MnCl2reduced NK activity. NK activity in cultures treated with MnCl2was also reduced upon removal of plastic adherent cells. However, restoration of enhanced NK activity by addition of adherent cells to nonadherent cells in the presence of MnCl2was not observed. Similar effects of NK activity were observed with polyinosinic‐polycytidylic acid (Poly I · C), a known interferon inducer and NK enhancer. The results demonstrate that murine splenic NK activity is enhanced in vitro by MnCl2and that this enhancement may be mediated by interferon induction. The results also suggest that in vitro enhancement of NK activity by MnCl2, as with Poly I · C, may require participation of an adherent cell population for NK augmentation.