Aspergillosis continues to be a devastating disease entity that results in significant mortality in immunosuppressed patients. Rapid diagnosis is often required to initiate appropriate therapy. Although the histopathologist may be able to visualize fungal organisms in tissue specimens, the histology of Aspergillus species may overlap with a variety of fungi, so diagnosis often relies on fungal cultures that can take weeks to complete. Recently, an in situ hybridization assay targeting Aspergillus 5S ribosomal RNA (rRNA) was reported. This assay proved to be useful when fungal cultures were negative or not performed but when fungi compatible with Aspergillus species were identified in tissue sections. That study was performed to compare the probe described in the previous study (5S-1 probe) with two other probes specific for Aspergillus. Two customly designed 21- and 23-base oligonucleotide probes complementary to 5S (5S-2 probe) and 18S (18S-1 probe) rRNA of Aspergillus were synthesized and labeled with multiple biotin moieties at the 3' termini. By GenBank analysis, the sequence of the 18S-1 probe was shown to have 90% to 100% homology to Aspergillus fumigatus group, Aspergillus flavus, Aspergillus niger, Aspergillus terreus, Aspergillus parasiticus, Aspergillus tamarii, and Aspergillus glaucus group; the 5S-2 probe was homologous to Aspergillus flavus, Aspergillus niger, Aspergillus nidulans, Aspergillus awamori, and Aspergillus wentii. In situ hybridization was performed on 43 cases of Aspergillus infection including 41 localized aspergillomas in the lung, brain, sinonasal tract, and ear, and 2 cases of invasive aspergillosis involving pleura and soft tissue of the scapular region. The results were compared with those obtained using a previously reported 5S-1 probe. In situ hybridization was positive in 38, 38, and 40 cases with the 5S-1, 5S-2, and 18S-1 probes, respectively. The 18S-1 probe was most useful because of a wider detection spectrum. In situ hybridization for Aspergillus rRNA provides a useful means for rapidly and accurately identifying Aspergillus in tissues and may be useful if fungal organisms suggestive of Aspergillus species are present but if cultures are negative or have not been performed.