The authors analyzed 25 paraffin-embedded lung biopsy specimens for mycobacterial DNA by the polymerase chain reaction (PCR) from patients with pulmonary mycobacterial infection demonstrated by acid-fast stain, culture, or both. DNA was extracted from 4 microM unstained paraffin sections by proteinase K digestion followed by freeze-fracturing and amplified by nested PCR with primers for the mycobacterial 65-kDa antigen gene. Mycobacterial DNA was detected in 7 of 7 wedge and 9 of 18 transbronchial biopsy specimens by PCR. Nested PCR with direct visualization on an agarose gel was as sensitive as Southern blot hybridization. Serial dilution studies demonstrated that nested PCR could detect DNA amplified from 4-8 acid-fast organisms from a paraffin section. Restriction enzyme digestion of the amplified PCR product differentiated Mycobacterium tuberculosis from Mycobacterium avium-intracellulare. Polymerase chain reaction can detect low numbers of acid-fast organisms in paraffin sections and confirm and presumptively speciate mycobacterial infection when cultures are negative or not obtained.