The clinical significance of positive bacterial cultures in chronic sinusitis is often difficult to assess. Contaminants from surface colonization of the sinus mucosa may be difficult to distinguish from true intramucosal or bone involvement. Furthermore, tissue Gram stains are frequently unable to demonstrate the presence of bacteria in tissue despite endoscopic evidence of active sinusitis. In situ hybridization (ISH) techniques using bacterial rRNA probes were applied to evaluate the presence of intramucosal and intraosseous bacteria in chronic sinusitis surgical specimens. A total of 22 specimens of chronically inflamed human ethmoid bone were evaluated by ISH and by Gram stain. In three specimens, ISH identified bacterial rRNA within sinus mucosa and mucin. Notably, in these three ISH-positive specimens, Gram stain was negative in two. No specimen showed evidence of bacterial rRNA within bone. These preliminary results suggest that in situ hybridization may be a useful adjunct to current methods of detecting microorganisms within chronically infected sinus tissue.