An in-situ hybridisation (ISH) technique to detect Aspergillus fumigatus in infected tissues was developed in which 568-bp, 333-bp and 154-bp PCR products of the alkaline proteinase gene were employed. Dot-blot hybridisation with the 568-bp probe on a membrane containing genomic DNA from several different fungi including A. flavus, A. niger, Penicillium spp., Mucor racemosus or Pseudallescheria boydii gave negative results. ISH was done on formalin-fixed, paraffin-embedded pulmonary tissues from rats infected with A. fumigatus and renal tissues from mice infected with A. fumigatus, A. flavus or A. niger. The 568-bp probe reacted strongly in ISH with both A. fumigatus and A. flavus, and weakly with A. niger. The 333-bp probe also reacted in ISH with A. fumigatus and A. flavus, although the intensity was weaker. However, in ISH with the 154-bp probe, there was no positive signal with any Aspergillus spp. These results demonstrate that A. fumigatus and A. flavus can be specifically detected in infected tissues by ISH with the 568-bp probe. This technique could be applicable to clinical specimens for molecular diagnosis of aspergillus infections.