At the Animal Health Service in the Netherlands faecal culture for M. paratuberculosis is performed on a relatively large scale. Routinely, faecal samples are decontaminated, and subsequently cultured on Lowenstein-Jenssen medium in the presence of mycobactin. Cultures are inspected every four weeks for up to 26. When suspected colonies were present, a Ziehl-Neelsen stain is performed. Ziehl-Neelsen positive cultures are at the moment confirmed using the IS900 PCR. We evaluated the confirmation results on 426 Ziehl-Neelsen positive cultures using the IS900 PCR. Only one out of 426 PCR reactions was negative without amplification of the internal control, apparently due to inhibition. However, 69 out of 425 (16 %) Ziehl-Neelsen positive cultures were negative in the IS900 PCR, whereas 356 Ziehl-Neelsen positive cultures were positive in the IS900 PCR. Subsequently, the mycobacterial status of 32 of the IS900 PCR negative suspensions was evaluated, using a PCR/crossblot technique. In 1out of 32 samples a positive result was obtained with a IS900 based M. paratuberculosis specific probe, indicating a false-negative result in the IS900 PCR. In all other suspensions the strongest reaction patterns were observed with either a probe against the M. avium-complex (in the absence of a reaction with a IS900 specific probe) or with a probe against M. phlei. However, in all crossblot patterns also other reactions were observed against other mycobacterial species, like M. intracellularis, M. gordii, and M. smegmatis, indicating the heterogeneous nature of these crude suspensions. In our opinion, these results once again underline, that Ziehl-Neelsen positive cultures should be positively identified as M. paratuberculosis, either by demonstrating the mycobactin dependency or by a specific IS900 PCR.