Faecal samples (40 g) were collected from 22 dairy cows. The cows were chosen because Mycobacterium paratuberculosis was detected in faecal cultures 6 to 8 months earlier. Each faecal sample was distributed over ten split-samples. Each split-sample was decontaminated by mixing 3 g faeces and 8 mL NaOH 4% in a mortar to create a homogeneous suspension. This suspension was put in a centrifuge tube and shaken for 15 min. After centrifugation during 15 min at 2500 rpm the supernatant was removed. To the pellet 5 mL oxalic acid (5%) and 0.1% malachitgreen were added. This mixture was shaken for 15 min, followed by centrifugation (15 min, 2500 rpm). The supernatant was removed and to the pellet 6 mL of an antibiotic mixture of Amphotericin and Neomycin was added, vortexed briefly and incubated for 18-24 h at room temperature ( 20-22 °C). Than 0.8 mL of the sample was distributed over 4 culture tubes with Lowenstein - Jensen medium. The inoculum was taken just above the settled pellet. The culture tubes were placed in an incubator at 37°C in slanted position for one night during which time some evaporation took place, after this the tubes were placed vertical while the caps were firmly fastened. The media were checked for typical colony forming units (CFU) of M. paratuberculosis after an incubation period of 6, 8, 12 and 16 weeks. The number of positive tubes after 6, 8, 12 and 16 weeks was respectively: 82 (9.3%), 236 (26.8%), 293 (33.2%) and 347 (39.8%). There were 13 split-samples with only one tube positive after 16 weeks. The number of positive split-samples after 6, 8, 12 and 16 weeks was respectively: 22 (10%), 87 (39.5%), 106 (48.2%) and 110 (50%). The distribution of the positive split-samples varied between 0 to 10 per cow-sample. After 16 weeks 7 cow-samples were negative in all ten of the split samples, 3 cow-samples resulted in only one positive split-sample, 1 cow-sample had three positive split-samples, 1 cow-sample had eight positive split-samples, 4 cow-samples had nine positive split samples and 6 cow-samples were positive in all ten split-samples. In two samples of these six heavy shedders all tubes were positive as early as 6 weeks incubation with numbers between 10 to 100 CFU per tube. It is concluded that M. paratuberculosis is not homogeneously distributed in faeces, but present in small clusters. Therefor from cows with low numbers of M. paratuberculosis in the faeces, faecal cultures may not necessarily give positive results.