A number of studies have substantiated the pivotal role of innate defense mechanisms in protection against invasive aspergillosis. However, experiments demonstrating increased resistance to lethal intravenous (i.v.) infection with Aspergillusfumigatus conidia in cortisone-treated or untreated mice preinfected with a sublethal dose of conidia and protection of turkeys inoculated subcutaneously with a killed A.fiumigatus germling vaccine against subsequent aerosol challenge led us to speculate that acquired immunity may also contribute to host defense against Aspergilus infection. Five-week-old male BALB/c mice were inoculated i.v. with 1.0 x 104 viable conidia or saline and challenged i.v. with 1.0 x 106 conidia after 7, 15, or 21 days. No protection against challenge was found after 7 days. However, significant and reproducible protection was observed after 15 and 21 days. Mortality was reduced from 90% in control mice to 53% in preinfected mice 40 days after challenge (P = 0.0002). Increased survival was correlated with decreased content of chitin in lungs, liver, and kidneys 4 and 7 days after challenge (P < 0.05). Mice were again inoculated with 1.0 x 104 conidia or saline, and after 21 days, 1.0 x 10' or 2.0 x 108 splenocytes were transferred to naive syngeneic recipients; 2.0 x 10' immune splenocytes conferred significant protection (P = 0.0001) against i.v. challenge with 1.0 x 106 conidia, and mortality decreased from 83 to 48% 40 days after challenge. Transfer of immune serum offered no protection despite the presence of antibody against a hyphal homogenate ofA.fmigatus, which was absent in the sera of control mice. Protection by immune splenocytes was maintained after selective depletion of T cells but was abolished after removal of plastic-adherent splenocytes. Adherent cells were characterized as macrophages by using morphological criteria, nonspecific esterase, and MAC-1 monoclonal antibody. Production of hydrogen peroxide by peritoneal and splenic macrophages from preinfected mice was the same as and lower than, respectively, that from uninfected controls. However, phagocytosis of conidia by peritoneal or splenic macrophages from mice preinfected i.v. or intratracheally was significantly increased after 2 and 3 h of coculture compared with that from uninfected animals, whereas in vitro killing of conidia by splenic macrophages was unaltered. Peritoneal or splenic macrophages from control or preinfected mice failed to kill hyphae in vitro. Killing of hyphae by polymorphonuclear leukocytes was not significantly different between mice preinfected i.v. and uninfected controls. Taken together, the results indicate that acquired immunity mediated by activated macrophages can be demonstrated in experimental murine aspergillosis. Although the mechanism is present biologically, its relevance against the invasive hyphal form ofA. fimigatus is doubtful.