Abstract A longitudinal study of sympathetic noradrenergic (NA) innervation of the spleen was carried out in 3-, 8-, 12-, 17-, 21-, and 27-month-old Fischer 344 (F344) rats using (i) fluorescence histochemistry for localization of norepinephrine (NE); (ii) immunocytochemistry (ICC) for localization of tyrosine hydroxylase (TH)-positive nerve fibers alone, and in combination with specific markers for T and B lymphocytes (OX19 and anti-μ respectively), and macrophages (ED3); and (iii) high-performance liquid chromatography with electrochemical detection for quantitation of NE. Fluorescence histochemistry revealed extensive loss of NA nerve fibers in all compartments of the spleen in 21- and 27-month-old rats. With single-label ICC, a decline in TH+ nerve fibers in all compartments of the spleen was observed by 17 months of age and became more severe with advancing age; these findings suggest that both the ratelimiting enzyme and the transmitter itself (NE) are depleted from sympathetic nerves in aged rat spleen. Double-label ICC demonstrated the loss of TH+ nerve fibers in spleen from 17-, 21-, and 27-month-old rats, and a parallel loss of OX19+ T lymphocytes and ED3+ macrophages in these cellular compartments. Neurochemical measurement of NE demonstrated a decline in NE per wet weight at 27 months of age. The age-related decline in NA innervation of spleen and in the density of specific populations of cells of the immune system (T lymphocytes and antigen-presenting ED3+ macrophages), that follow remarkably similar time courses, supports functional evidence for dynamic interactions between the immune system and NA sympathetic nerves in the spleen, and further suggests a causal relationship between these age-related phenomena, i.e., that age-related immunosenescence promotes sympathetic denervation of the spleen which further compromises immune function. This hypothesis, however, requires further testing.