USDA regulations have stated that an organism-based test (culture) is the official test for determining the infective status of an animal for Johne's Disease. Recent method evaluation tests performed for laboratory approval for the United States Voluntary Johne's Disease Herd Status Program (USVJDHSP) indicate multiple culture methods were being used in the United States. The yearly evaluations have indicated that there were a wide range of sensitivities associated with the different culture methods. The National Veterinary Services Laboratories (NVSL) have been requested to establish a standardized protocol for detecting Mycobacterium avium subspecies paratuberculosis (MAP) in fecal samples which is reproducible and has a known sensitivity. The NVSL have also been requested to establish the criteria for well-characterized bovine fecal panels for use in organism-based detection procedures and methods evaluation. These panels will be used to validate different diagnostic procedures, including serological assays and USDA licensed diagnostic kits used for Johne's Disease detection. Based on the results of the last 3 years of proficiency tests, several methods have been chosen for further evaluation. These methods included sedimentation, centrifugation and liquid culture procedures which have been used by multiple laboratories. Preliminary evaluation based on proficiency test results indicate that centrifugation methods are more sensitive than sedimentation methods and liquid culture methods are faster than either sedimentation or centrifugation methods using Herrold's Egg Yolk (HEY) media with mycobactin. Varied growth performances in the solid media used with different culture methods were also noted during the last 2 check tests. There are now 2 commercial sources of HEY media with mycobactin available in the United States which will be evaluated along with in-house media for growth performance.