The high sensitivity of PCR compared to the difficulties of fecal culture in sheep prompted the development of PCR protocols for detection of Mycobacterium avium subsp. paratuberculosis DNA in sheep feces. Although the PCR itself is well developed, and does not pose large technical problems, concentrating the bacteria from samples that may contain low numbers of bacilli using practical methods is still the main difficulty for the use of this technique. In this study, we describe an extraction protocol for the concentration and purification of M. avium subsp. paratuberculosis DNA from fecal samples and we compare it with other methods. The diagnostic performance of the freeze-boiling method was evaluated using a reference method [Vet. Rec. 134 (1994) 95] on fecal samples from a group of selected sheep from different flocks of known individual serological, pathological, and cultural paratuberculosis status. Using, as a reference, a combination of results in those conventional methods, the freeze-boiling PCR protocol showed a sensitivity of 94.1%, and a specificity of 92.3%.