Natural killer (NK) cell activity has been postulated to be an immunologic link between beta-carotene and cancer prevention. In a cross-sectional, placebo-controlled, double-blind study we examined the effect of 10-12 y of beta-carotene supplementation (50 mg on alternate days) on NK cell activity in 59 (38 middle-aged men, 51-64 y; 21 elderly men, 65-86 y) Boston area participants in the Physicians' Health Study. No significant difference was seen in NK cell activity due to beta-carotene supplementation in the middle-aged group. The elderly men had significantly lower NK cell activity than the middle-aged men; however, there was no age-associated difference in NK cell activity in men supplemented with beta-carotene. beta-carotene-supplemented elderly men had significantly greater NK cell activity than elderly men receiving placebo. The reason for this is unknown; however, it was not due to an increase in the percentage of NK cells, nor to an increase in interleukin 2 (IL-2) receptor expression, nor to IL-2 production. beta-carotene may be acting directly on one or more of the lytic stages of NK cell cytotoxicity, or on NK cell activity-enhancing cytokines other than IL-2, such as IL-12. Our results show that long-term beta-carotene supplementation enhances NK cell activity in elderly men, which may be beneficial for viral and tumoral surveillance.