Faecal samples from Danish cattle were investigated for the presence of Mycobacterium avium subsp. paratuberculosis by 3 laboratories by their routine cultivation procedures. Out of 30 samples of which 14 previously had been found culture positive, the 3 laboratories (named A, B and C) found 12, 6 and 0 positive respectively by their direct standard culture methods. One laboratory (A) also performed direct PCR on the samples and found 13 positive. Two laboratories (A and C) also performed culture combined with PCR and found 14 and 10 positive respectively. These findings show that laboratories certified to perform cultivation for Mycobacterium avium subsp. paratuberculosis need to evaluate and renew their diagnostic methods regularly to ensure the best results.