In a cross-sectional study on milk samples from 1155 cows from 22 Danish dairy herds, selected risk factors for paratuberculosis were identified. The diagnostic procedure used was an indirect enzyme-linked immuno-sorbent assay (ELISA) to detect antibodies against Mycobacterium avium subsp. paratuberculosis. A sample was considered test-positive if it had a corrected optical density >/=0. 025 (test sensitivity 71.4% and test specificity 89.7%). Of the 1155 samples, 8.8% (102/1155) were test-positive, and 19 out of the 22 dairy herds had >/=1 test-positive cows. The significant risk factors in a multiple logistic regression analysis were: Jersey versus large breeds, high parity versus low parity, the first month after calving versus other months of lactation, and a large herd size compared to a small herd size. The highest probability (37-38%) of a positive test was observed among older cows (parity >4) and tested within the first month after calving (irrespective of breed). The lowest probability (2%) of a positive test-result was observed among first parity, large-breed cows tested before calving or later than one month after.