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Three commercially available assays, designed to specifically detect the presence of Mycobacterium avium subsp. paratuberculosis (MAP) in fecal samples by IS900-PCR, were compared with a conventional culture method. Fecal samples from 100 dairy cows were tested. Fifty-four (67.5%) of 80 culture-positive samples were positive for an assay that detects MAP DNA by dot spot hybridization of polymerase chain reaction products (kit A), 48 (60%) were positive by an assay using ethidium bromide staining for agar gel visualization of amplification products (kit B), and 49 (61.3%) were positive by an assay in which amplified products are detected by a colorimetric detection system (kit C). Relative sensitivity of all tests increased in proportion to the presence of MAP in fecal samples. Specificity was 100% based on results from 20 culture-negative samples from an MAP-free herd.