Diagnosis of paratuberculosis using the IDEXX DNA probe test and three methods for cultivation of M. paratuberculosis from fecal specimens were compared. Twenty-one of 170 fecal specimens were DNA probe test positive, whereas 35 specimens were culture positive by one or more of the methods evaluated. Four specimens were DNA probe positive but culture negative. The probe test detected M. paratuberculosis DNA in 62.9% of specimens positive by a sedimentation method, 56.6% of those positive by a centrifugation method, and 65.4% of those postive by the Cornell culture method. Generally, the probe test was positive for cattle shedding at least 10⁴M. paratuberculosis organisms per gram of feces. It was possible to identify cattle shedding the greatest number of organisms in 3 days using the probe test compared with a minimum of 6 weeks required for positive culture results. After 12 weeks incubation, the centrifugation method resulted in the most isolations of M. paratuberculosis and the most contamination of culture medium. Contamination was best controlled using the Cornell culture method. The sedimentation method was the least time-consuming and yielded results similar to the other two methods.