Dietary folate deficiency enhances, whereas folate supplementation suppresses, the development of several cancers. This study investigated the effect of folate deficiency on natural killer cell (NK)-mediated cytotoxicity, which is important in immune surveillance against tumor cells. In Experiment 1, severe folate deficiency was induced in rats by feeding an amino acid-defined diet containing 0 mg folate and 10 g succinylsulfathiazole/kg diet. Control and folate-supplemented rats were fed the same diet containing 2 (basal requirement) and 8 mg folate/kg diet, respectively. Severe folate deficiency at the end of wk 5 was associated with 20% growth retardation, a 60% reduction in lymphocyte counts and significantly impaired NK-mediated cytotoxicity compared with the control and folate-supplemented groups (P < 0.02). The lesser degree of severe folate deficiency achieved by wk 4 was not associated with impaired NK-mediated cytotoxicity. Folate supplementation at 4x the basal requirement did not significantly enhance NK-mediated cytotoxicity at either time point. In Experiment 2, moderate folate deficiency was induced in rats by feeding the same diet without succinylsulfathiazole. NK-mediated cytotoxicity in the moderately folate-deficient rats (without growth retardation or lymphopenia) was not significantly different from that in controls. Although severe folate deficiency may have adverse effects on NK-mediated cytotoxicity, moderate folate deficiency, a degree of depletion associated with an increased risk of several cancers, appears not to affect NK-mediated cytotoxicity in rats. Furthermore, a modest level of folate supplementation above the basal requirement does not enhance NK-mediated cytotoxicity. These data collectively suggest that NK-mediated cytotoxicity is not a likely mechanism by which folate status modulates carcinogenesis.