In recent years nontuberculous mycobacteria (NTM) have reportedly been on the increase caused by opportunistic infections. The bacteria within the M. chelonae complex (MCC; M. chelonae, M. abscessus and M. immunogenum) have been under investigation as to their role with respect to hypersensitivity pneumonitis in automobile workers and other occupational workers. Detection of these bacteria in metal working fluids (MWFs) has traditionally been by culture complemented by PCR and restriction digests of products to discriminate at the species level. Real-time PCR assays have to date only been able to distinguish to the genus level using a SYBR Green labelling assay. Here we describe a novel quantitative real-time 5-Nuclease (Taqman) PCR technique was developed to specifically detect M. immunogenum. The specific primers and Taqman probe were designed to amplify a 60 bp region of the rpoB gene. Specificity for M. immunogenum was confirmed when tested against close mycobacterial relatives and Rhodocccous equi strains. The sensitivity of detection of M. immunogenum genomic DNA was shown to be approximately 9 fg (2 cell equivalents). The assay was successfully applied to the detection of M. immunogenum in industrial metal working fluids from the U.K. and the U.S.A demonstrating its applicability to industry. In particular, this method permits the detection of M. immunogenum and to clarify the possible link to hypersensitive pneumonitis, which is under investigation since more than 15 years.