By comparing natural immunity to Aspergillus fumigatus (AF) in vivo with the action of human or mouse phagocytes against AF in vitro, we delineated two sequential lines of defense against AF. The first line of defense was formed by macrophages and directed against spores. Macrophages prevented germination and killed spores in vitro and rapidly eradicated conidia in vivo, even in neutropenic and athymic mice. The second was the neutrophilic granulocyte (PMN), which protected against the hyphal form of AF. Human and mouse PMN killed mycelia in vitro. Normal, but not neutropenic mice, stopped hyphal growth, and eradicated mycelia. Either line of defense acting alone protected mice from high challenge doses. Natural immunity collapsed only when both the reticuloendothelial system and PMN were impaired. These findings are in keeping with the clinical observation that high doses of cortisone and neutropenia are the main risk factors for invasive aspergillosis. Cortisone inhibited the conidiacidal activity of mouse macrophages in vivo and of human or mouse mononuclear phagocytes in vitro. Cortisone damaged this first line of defense directly and not through the influence of T lymphocytes or other systems modifying macrophage function as shown in athymic mice and in vitro. In addition, daily high doses of cortisone in mice reduced the mobilization of PMN so that the second line of defense was also impaired. Thus, cortisone can break down natural resistance on its own. Myelosuppression rendered mice susceptible only when the first line of defense was overpowered by high challenge doses, by activated spores that cannot be killed by macrophages, or by cortisone suppression of the conidiacidal activity of macrophages. The host, thus, can call upon two independent phagocytic cell lines that form graded defense systems against aspergillus. These lines of defense function in the absence of a specific immune response, which seems superfluous in the control and elimination of this fungus.