Polymerase chain reaction (PCR) based on amplification of the IS900 sequence has been reported to be a rapid test for detecting Mycobacterium avium subsp. paratuberculosis (MAP) in feces. However, fecal culture is still recognized to be the most sensitive technique as the confirmative diagnostic test of MAP, because of the problems associated with the low numbers of MAP in feces during early infection and the resistance of the bacterial cell wall to lysis, together with PCR-inhibitory substances present in fecal samples. We have developed a novel method of DNA preparation from fecal samples, which is able to effectively extract bacterial DNA and eliminate inhibitory substances. In this method, DNA was extracted using micro beads in lysis buffer, then purified using organic solvents. We compared the usefulness of our DNA preparation method for MAP-PCR test with the classical heat extraction method. Fecal samples of known MAC status were obtained, which contained a lot of MAP organisms. DNA samples were prepared by both methods from fecal residues, which had previously been subjected to a single heat extraction. IS900-specific PCR products were detected from fecal DNA fraction prepared by our method, but not detected from samples prepared by additional heat treatment. These results show some MAP organisms are excreted into feces in the conditions exhibiting a highly resistance against the heat treatment alone. In addition, we have developed a reagent cocktail for PCR (Ampdirect(r)) that can neutralize the inhibitory effects of substances in biological samples on DNA amplification. Using our DNA preparation method and Ampdirect(r) reagent, IS900-specific PCR products were detected in all four fecal samples from experimentally infected calves, that showed none or a few formation of MAP colony in fecal culture. The combination of our DNA preparation method with PCR using Ampdirect(r) thus offers a detection method for MAP that is more sensitive than the fecal culture and much more rapid.