Since certain functions mediated by nitric oxide (NO) decline with age, the age dependence of NO production by macrophages from BALB/c mice was investigated. Lipopolysaccharide-, peptidoglycan-polysaccharide-, or interferon-γ-stimulated splenic and peritoneal macrophages from young (1 month old), middle-aged (4-5 months old), and old (6-20 months old) BALB/c mice showed a progressive and marked decline in NO production. This age- related decline in inducible NO extended to C57/BL6 and CB6F1 mice. mRNA for inducible NO synthase (iNOS), the enzyme responsible for inducible NO production by macrophages, also declined with age. Importantly, the reduced NO production by macrophages from old mice could be up-regulated by pretreating the mice with either cholera toxin or concanavalin A. These findings indicate that reduced production of NO by murine macrophages correlates directly with advancing age, likely due to deficient signals or signal transduction responsible for iNnS mRNA and protein generation.