There is a need for rapid and sensitive detection of Mycobacterium tuberculosis in tissue specimens. A polymerase chain reaction (PCR)-based assay for the diagnosis of tuberculosis was evaluated in 60 formalin-fixed tissue specimens, the target for the amplification being a segment of IS6110 in the M. tuberculosis chromosome. Of the 60 formalin-fixed, paraffin-embedded tissue specimens studied, 57 showed granulomatous inflammation and 53 had been cultured for mycobacteria; 10 were positive for M. tuberculosis and three were positive for other mycobacteria. Of 60 samples, 15 showed acid-fast bacilli on special staining. When done comparatively on a positive culture for M. tuberculosis, PCR for M. tuberculosis DNA in 60 tissue samples was 100% sensitive and 93% specific, having a positive predictive value of 76.9% and negative predictive value of 100%. PCR for M. tuberculosis DNA done on tissue samples was positive for 14 of 19 patients who had a clinical diagnosis of tuberculosis, negative for all six patients with nontuberculous mycobacterial infections, and negative for all 33 patients who had a diagnosis of a disease other than mycobacterial infection. When compared with the clinical diagnosis of tuberculosis, PCR for M. tuberculosis DNA in these patients' tissues was 73.6% sensitive and 100% specific, having a positive predictive value of 100% and negative predictive value of 88.6%. These data indicate that PCR amplification is useful for detecting M. tuberculosis DNA in formalin-fixed tissue specimens, and that it can be used to increase diagnostic accuracy in patients who have perplexing diagnostic problems associated with a granulomatous tissue response.