PCR as diagnostic method for detecting Mptb offers an attractive alternative for the traditional cultural methods combining both rapidity and specificity. Allthough the Mptb specific IS900 sequence offers a specific PCR target, reports on false positive IS900 PCR results implicate the need for product confirmation by hybridization. The TaqMan technology combines the benefits of PCR with confirmation by hybridization. Furthermore the TaqMan technology enables quantitative PCR and high through-put screening. The aim of this study was to develop a rationalized approach for isolating and detecting Mptb from faecal samples taking into account the specificity of the primers and probe for IS900 and monitoring PCR inhibition by spiking samples with a mimic DNA template and subsequent detection with TaqMan technology. The extraction of DNA in our method avoids organic extraction by capturing Mptb cells from fecal samples using anti-Mptb directed monoclonal antibodies coated to magnetic beads. Faeces from non-infected cows (repeatedly tested by culturing) were spiked with Map cells and isolated by immunomagnetic capture. An average of 10 cells per gram of faeces could be detected by using this technique. Inhibition of the PCR reaction was very low, especially when compared to detection methods based on DNA binding to a silica-gel column. Furthermore, 34 faecal samples from a known cultural status (19 positive,15 negative) were tested using the PCR method showing a good agreement with the culturing data as is expressed by a kappa value for agreement of 0.76.