To evaluate the role of mycobacterial infection in the pathogenesis of sarcoidosis, several groups have attempted to identify mycobacterial DNA in clinical samples from these patients by polymerase chain reaction (PCR), but widely divergent results have been obtained. It has been suggested that differences in the sensitivity of the procedures used may explain these discrepant results. To test this possibility, the presence of mycobacterial DNA was sought in biopsies from patients with sarcoidosis using sequence capture-PCR, a procedure that is 100-fold more sensitive in detecting mycobacterial DNA in paucibacillary samples than standard PCR protocols. Using this approach, DNA corresponding to two different sequences specific for organisms of the Mycobacterium tuberculosis complex (the 1S6110 insertion element and the DR region) could not be detected in any of the 15 biopsies from patients with sarcoidosis, whereas a high proportion of positive results was obtained for tissue biopsies and other clinical samples from patients with active tuberculosis, including samples that were smear-negative/culture-positive and smear-negative/culture-negative. These results support prior studies suggesting that M. tuberculosis does not play a pathogenic role in sarcoidosis in most patients.