Cord factor (trehalose 6,6'-dimycolate: TDM) is a well-known toxic glycolipid in Mycobacterium tuberculosis. We isolated various mycoloyl glycolipids from Nocardia asteroides 23,167 and related species which are closely related taxonomically to Mycobacterium. Since Nocardia is also an opportunistic pathogen co-infected with HIV, we examined in vivo granuloma formation and the in vitro macrophage activation in mice. We found that cord factor (TDM) and glucose monomycolate (GM) from Nocardia asteroides and Rhodococcus species with shorter chain mycolic acids also exhibited distinctive granuloma-forming activity in lungs, spleen and liver in mice and in vitro induction of prostaglandin E2 (PGE2) and interleukin 1 (IL-1) synthesis. We also found that mycoloyl glycolipids possessing trehalose or glucose as a carbohydrate moiety exhibited immunomodulatory activity, and that mycoloyl glycolipids with longer chain mycolic acids exhibited stronger activity in mice than did those with shorter chain mycolic acids. The mycoloyl glycolipids from Nocardia asteroides directly activated macrophages. Stimulation of above concentration with 0.16 microgram/ml of TDM or 0.8 microgram/ml of GM markedly enhanced production of PGE2 by mouse peritoneal macrophages. At higher concentrations above 100 micrograms/ml of TDM or 500 micrograms/ml of GM, IL-1 release was also enhanced.