Aims: To examine the effects of ammonium feeding on the production of cordycepin (3'-deoxyadenosine, a nucleoside analogue) and exopolysaccharides (EPS) in mycelial culture of a new Cordyceps sinensis fungus Cs-HK1. Methods and Results: Cs-HK1 fungus was cultivated in a liquid medium containing glucose, yeast extract, peptone and a few major inorganic salts. NH4Cl was fed to the mycelial culture at various concentrations from 5 to 40 mmol l(-1) on day 3 (during exponential phase). NH4Cl, fed at 10 mmol l(-1), stimulated the cordycepin production most significantly, with nearly fourfold increase in the cordycepin content of mycelia (from 28.5 to 117 mu g g(-1)), and also increased the EPS production by 40% (from 2.6 to 3.7 g l(-1)). The ammonium feeding had a slightly positive effect at 5-10 mmol l(-1), but a negative effect at higher concentrations on the mycelium growth. Ammonium feeding also caused a sharp drop of the medium pH, owing perhaps to the uptake of NH3 and the release of H+ by the fungal cells. Conclusions: Ammonium feeding to the mycelial culture of Cs-HK1 fungus enhanced the intracellular cordycepin accumulation and the EPS production. The enhanced cordycepin production may be attributed to the uptake of ammonia for nucleoside synthesis, and the enhanced EPS to the increased uptake of glucose for EPS biosynthesis. Significance and Impact of the Study: It is useful for the production of bioactive metabolites and for understanding ammonium metabolism and its relationship to the biosynthesis of nucleosides in a precious medicinal fungus.