The spores of Aspergillus fumigatus have a survival advantage over other respirable fungal spores in the lung, leading to a number of lung diseases associated with this fungus. We have hypothesized that a component on the spore surface can inhibit the activation of alveolar macrophages, known to play an essential role in immune regulation in the lung. A diffusible product from the spores of A. fumigatus (AfD) inhibited the production of tumor necrosis factor alpha (TNF alpha) protein by alveolar macrophages in an enzyme-linked immunosorbent assay. Using a semiquantitative reverse transcription-polymerase chain reaction, we also demonstrated a potent inhibitory effect of AfD on the production of proinflammatory cytokine transcripts in rat alveolar macrophages. The inhibition occurred at the level of transcription, with AfD inhibiting the synthesis of TNF alpha-and interleukin 6 (IL-6)-specific mRNA transcripts. No effect was seen on the synthesis of interleukin 1 beta (IL-1 beta) cytokine transcripts or on the expression of the housekeeping gene beta-actin. Furthermore, AfD specifically inhibited the activation of nuclear transcription factors NF-kappa B and AP-1, both of which are required for the coordinate upregulation of transcription of the proinflammatory cytokines TNF alpha, IL-1 beta, and IL-6. We conclude that AfD can inhibit normal alveolar macrophage responses by selectively inhibiting the production of key inflammatory cytokines, and that the mechanism of inhibition is primarily at the level of transcriptional activation.