The purpose of the study was to determine the distribution of Mycobacterium avium ssp. paratuberculosis (MAP) across the main milk and colostrum fractions (cream, curd, and whey). Raw milk and colostrum were inoculated with 1 of 2 MAP strains, ATCC 19698 or S-23, yielding initial concentrations of 10(6) to 10(7) cfu/mL. After fractionation, for milk as well as for colostrum, 80 to 90% of the recovered MAP cells were found in the curd fraction and 10 to 20% in the cream fraction. Total MAP colony counts in milk whey were 4 to 5 log(10) units lower than colony counts of inoculated milk. In colostrum, colony counts were 2 to 3 log(10) units lower in whey than in inoculated colostrum. Because of the slow growth of MAP and to proceed more smoothly with set-up and optimization of the method, luminescent MAP strains were used. The high correlation coefficient (r=0.960) between colony counts and luminescence measurements showed that the use of luminescent MAP strains during method development was plausible.