Microscopic examination of tissue sections of mycobacterial lesions frequently results in few or no bacilli seen, even if the lesions appear active histologically. This might be due to the effects of the fixative fluid and/or organic solvent, both of which are conventionally used to make tissue sections for histopathology, on the acid-fast staining of bacteria. The present study was performed to examine how formalin and xylene lower the sensitivity of acid-fast staining for Mycobacterium tuberculosis and to clarify the meaning of the staining result in tissue sections. Microscopic observation of mycobacteria smeared on glass slides revealed that both of these agents greatly reduced the sensitivity of acid-fast staining. Moreover, the number of bacilli was calculated in 30 samples of paraffin-embedded granulomatous lesions using acid-fast microscopy and real-time polymerase chain reaction. The numbers of bacilli present that were estimated by real-time polymerase chain reaction were considerably higher than those counted with a microscope. These results suggest that the bacilli are frequently missed or underestimated with acid-fast microscopy on formalin-fixed, paraffin-embedded tissue.