Although secreted proteins of pathogenic microorganisms often represent potential virulence factors, so far only limited information has been available on the proteins secreted by Aspergillus fumigatus. We therefore analysed supernatant proteins after growth in different media. In serum-free cell culture medium A. fumigatus growth was limited and no protein secretion was detectable, whereas distinct protein patterns were detectable after growth in either aspergillus minimal medium (AMM) or the more complex yeast glucose medium (YG). The three major proteins secreted under these conditions were identified as the ribotoxin mitogillin, a chitosanase and the aspergillopepsin i. Mitogillin and chitosanase were secreted in AMM, whereas aspergillopepsin i was especially prominent after growth in YG. When the AMM cultures reached stationary phase, seven additional major proteins were detectable. Two of them were identified as the chitinase chiB1 and a β(1–3) endoglucanase. Conditioned medium containing mitogillin and chitosanase did not have a detectable cytotoxic effect on A549 and Vero cells. Using recombinant mitogillin and chitosanase we detected anti-chitosanase and anti-mitogillin antibodies in sera of patients suffering from invasive aspergillosis or aspergilloma, but not in control sera of healthy individuals. This suggests that chitosanase, like mitogillin, is expressed during infection and might therefore be of diagnostic relevance.