The etiology of sarcoidosis is unknown, but mycobacteria have been considered as a possible etiologic agent. The authors used the polymerase chain reaction (PCR) to search for mycobacterial DNA in paraffin-embedded granulomatous tissues from patients with sarcoidosis. The target sequence used for PCR amplification is a 383-base pair segment of the gene encoding the 65 kD mycobacterial surface antigen. This assay can detect Mycobacterium tuberculosis and atypical mycobacteria in archival material. Its sensitivity, which is superior to Ziehl-Nielsen staining for acid-fast bacilli, is 1 bacterium per 2500 cells. Ten sarcoidosis blocks and 10 normal controls were negative with mycobacterial PCR but positive with beta-actin PCR, indicating the presence of amplifiable DNA. Mycobacterial PCR gave positive results for six acid-fast bacilli stain/culture-positive blocks from patients with tuberculosis. These results indicate that sarcoidosis probably does not represent an active mycobacterial infection. These data also suggest that mycobacterial PCR is helpful in differentiating tuberculosis and sarcoidosis.