Polymerase chain reaction (PCR) amplification of IS900 gene is recognised as a rapid method for the direct detection of Mycobacterium paratuberculosis from faecal samples. However, because of the problems associated with inhibitory activity of faeces, organisms or DNA templates have to be purified from faeces. These sample preparation steps are laborious and time consuming to perform PCR with large number of samples. Using the faecal samples taken from experimentally infected calves and artificially seeded faecal samples, several methods were evaluated for their ability to reduce PCR inhibitory activity of faeces. A nested PCR reported by Collins et al. was employed for the amplification of IS900 gene fragment, and the amplified products were electrophoresed on 2.5% agarose gels and were stained ethidium bromide. It was found that this inhibitory activity of faeces could be reduced by addition of Ampdirect( (Shimadzu, Japan) that neutralise PCR amplification inhibitors in blood and faeces. Furthermore, PCR amplifiable DNA could be prepared by using InstaGene( Matrix (Bio-Rad, USA) with simple procedures. In testing artificially seeded faecal samples, PCR product was detected at a concentration of 1.25% to 0.3% of faeces by addition of Ampdirect, whereas it had to be diluted 8 to 16 times more for PCR without Ampdirect to get positive reaction. Some of faecal samples from experimentally infected calves that were negative in PCR without Ampdirect showed strongly positive reaction with PCR using Ampdirect. Thus, the combination of DNA extraction with InstaGene Matrix and Ampdirect-PCR might be a rapid and simple method for the direct detection of M. paratuberculosis in faeces.