The diagnosis of mycobacterial infection depends on the Ziehl–Neelsen (ZN) stain, which detects mycobacteria because of their characteristic acid‐fast cell wall composition and structure. The histological diagnosis of tuberculosis (TB) comprises various aspects: (1) sensitive detection of mycobacteria; (2) precise localization of mycobacteria in the context of granulomatous lesions; (3) ‘staging’ of disease according to mycobacterial spread and granulomatous tissue integrity. Thus, detection of minute numbers of acid‐fast bacteria in tissue specimens is critical. The conventional ZN stain fails to identify mycobacteria in numbers less than 10⁴ per ml. Hence many infections evade diagnosis. PCR is highly sensitive, but allows neither localization within tissues nor staging of mycobacterial disease, and positive findings frequently do not correlate with disease. In this study, an anti‐Mycobacterium bovis bacille Calmette–Guérin polyclonal antiserum (pAbBCG) was used to improve immunostaining, which was compared to the ZN stain in histological samples. Screening of tissue samples including lungs, pleural lesions, lymph nodes, bone marrow, and skin for mycobacterial infection revealed that pAbBCG staining detects infected macrophages harbouring intracellular mycobacteria or mycobacterial material as well as free mycobacteria that are present at low abundance and not detected by the ZN stain. The positive pAbBCG staining results were confirmed either by PCR analysis of microdissected stained tissue or by culture from tissue. This immunostaining approach allows precise localization of the pathogen in infected tissue. Copyright © 2005 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.