Aims: To develop a fast and sensitive protocol for detection of Mycobacterium avium subsp. paratuberculosis (MAP) in bovine semen and to make a critical evaluation of the analytical sensitivity. Methods and Results: Processed semen was spiked with known amounts of MAP. Semen from different bulls as Well as semen of different dilutions was tested. The samples were treated with lysing agents and beadbeating and the DNA was extracted with phenol and chloroform. Real-time PCR With a fluorescent probe targeting the insertion element IS900 detected as few as 10 organisms per sample of 100 mu l semen. PCR-inhibition was monitored by inclusion of an internal control. Pre-treatment with immunomagnetic separation was also evaluated, but was not shown to improve the overall sensitivity. Conclusions: Real-time PCR is a sensitive method for detection of MAP ill bovine semen. Lysis by mechanical disruption followed by phenol and chloroform extraction efficiently isolated DNA and removed PCR-inhibitors. Significance and Impact of the Study: The high sensitivity of the applied method allows reliable testing of bovine semen used for artificial insemination to Prevent the spread of Johne's disease, caused by MAP.