Serum samples from 20 cows with spontaneously acquired systemic aspergillosis and serum collected consecutively from a cow with experimentally induced aspergillosis were assayed for antibodies against Aspergillus fumigatus in immunoblotting and enzyme-linked immunosorbent assays. Purified antigens (18 kDa protein and 20 kDa galactomannan) from A. fumigatus as well as complex preparations of metabolic and somatic antigens were used. Antibodies against A. fumigatus were found in both infected and non-infected cows. High titers and especially an increase in titer in consecutive samples signified infection. Reactivity with the 22 and 32 kDa antigens in preparations of exocellular antigens and the 19, 20, and 39 kDa antigens from intracellular antigens was seen with sera from infected cows only, and is therefore indicative of infection.