Objectives - To compare sensitivity of several methods of bacteriologic culture of pooled bovine fecal samples for detection of Mycobacterium paratuberculosis and evaluate homogeneity in number of M paratuberculosis in pooled fecal samples. Sample Population - Feces from 10 dairy cows that shed M paratuberculosis at various concentrations and 1 dairy cow known to be free of infection with M paratuberculosis. Procedure - 5 fecal pooling methods, 2 culture methods, and 2 pool sizes were evaluated. Each pooled sample contained 1 infected sample and 4 or 9 uninfected samples. Results - Sensitivity of detection of M paratuberculosis was greater with smaller pool size (5 vs 10 samples/pool). Detection sensitivity was also associated with concentration of bacteria in the infected sample. Results indicated that, compared with concurrent bacterial culture of individual infected samples, 37 to 44% of pooled samples with low bacterial concentrations yielded positive culture results and 94% of pooled samples with high bacterial concentrations yielded positive results. Conclusions and Clinical Relevance - Bacteriologic culture of pooled fecal samples may provide a valid and cost-effective method of detecting M paratuberculosis infection in cattle herds.