Arginine supplementation enhances in vitro lymphocyte proliferation in healthy adult humans and in rodent models. Studies examining the effect of arginine supplementation on in vivo immune responses are lacking. The purpose of this study was to determine whether arginine supplementation could enhance in vivo immune responses in adult mice and reverse known age-associated alterations in immune function of young and aged mice. Mice (1, 10 and 33 mo old) were fed a 2% arginine or an isonitrogenous diet for 2 wk. Delayed-type hypersensitivity to 2,4-dinitrofluorobenzene-challenged ears and changes in popliteal lymph node weights to injected sheep red blood cells were measured. The mean percentage of increase in ear thickness in challenged vs. unchallenged ears was 27, 35 and 24% with arginine supplementation and 7, 12 and 0% with the isonitrogenous diet in the 1-, 10- and 33-mo-old mice, respectively (P </= 0.05 for each age). Across all ages, the mean differences in popliteal lymph node weights were 1.9 +/- 0.3 vs. 1.0 +/- 0.3 mg with the arginine and isonitrogenous diets, respectively (P </= 0. 05). Only the 33-mo-old mice fed the isonitrogenous diet did not respond to this immune challenge. These findings suggest that arginine supplementation may enhance in vivo immune responses and/or reverse age-associated changes.