The suitability of a radiometric culture medium consisting of BACTEC 12B with PANTA PLUS, mycobactin J, and egg yolk was evaluated for detection of Mycobacterium paratuberculosis in feces, mesenteric lymph nodes, and intestinal walls from cattle, sheep, and goats. In addition, a simple method that would enable the rapid identification of Mycobacterium paratuberculosis by IS900 PCR in the primary cultures was sought so that subculture to secondary egg-free radiometric medium could be avoided. An ethanol extraction followed by differential centrifugation was used to separate M. paratuberculosis from PCR inhibitors in the primary culture. PCR was then undertaken with the pellet, after boiling to lyse the mycobacteria; if this test was negative, the DNA in the lysate was purified with guanidine thiocyanate and silica. Cultures of feces, ilea, and mesenteric lymph nodes from cattle, sheep, and goats known to have or suspected of having Johne's disease yielded positive PCR results 1 to 7 weeks after inoculation. Similar results were obtained with soil and pasture samples that had been spiked with M. paratuberculosis. The results suggested that radiometric culture was more sensitive than histopathology in detecting M. paratuberculosis infection in sheep and goats and more sensitive than culture on Herrold's egg yolk medium for the detection of the infection in cattle. Of 259 individual PCR tests with samples from cultures with growth indices of > or = 10,237 (91.5%) were positive, with only 28 (11.8%) requiring both ethanol and silica preparation to yield a positive result. Of the 22 negative PCR results for samples from cultures with growth indices of > or = 10, 18 were for samples from cultures that had only just developed evidence of growth. PCR-positive cultures tended to remain PCR positive over successive weeks. Flexibility in the timing of the sampling for PCR is thus possible, facilitating batch processing of samples in large-scale disease control programs for ruminants.