Confirmation of PTB infection has been hampered by both the lack of sensitivity of the currently available methods for Map isolation and the long time required for results. A promising method is the use of PCR in fecal samples having the advantage of providing rapid results and similar sensitivity as culture. Several PCR protocols based on the detection of the species-specific IS900 have been published. Sensitivity and specificity of PCR is increased by using nested PCRs. Additionally, cross-contamination between samples is prevented by a one tube nested (OTN) PCR since both amplification cycles take place in a single closed lid tube not opened until both amplification cycles are completed. In this study we optimized a OTN PCR and DNA extraction procedure from bovine feces for detection of Map in spiked fecal samples. This PCR will be run in parallel with traditional culture methods for confirmation of disease status in a study involving the evaluation of tests based on the detection of cellular and humoral immunity in young calves. Fecal samples were spiked with Map strain 19698 (final concentration: 1000, 250, 125, 62, and 31 cells/gram). DNA extraction was performed by combining freeze and thaw cycles with a commercial DNA extraction kit (Roche). Our OTN PCR utilizes a unique combination of those primers published by Englund et al. (1999) and IS900 is the target sequence. Extraction and OTN PCR were repeated 8 times/cell concentration. Obtained results of this study are: The OTN PCR detected as low as 62 cells/gram of feces in 87.5% of the spiked samples. Lower cell concentrations (31 cells/gram) in spiked feces were detected inconsistently, and in only 25% of the samples. The currently available culture methods provide a detection limit between 10 to 100 viable Map cells/gram of feces. Therefore, this extraction and PCR procedures demonstrate sensitivity levels in spiked fecal samples similar to culture.