Paratuberculosis is a chronic, infectious disease of ruminants that entails a serious concern for the cattle industry. One of the main issues relates to the efficiency of diagnosis of subclinically infected animals. The objective of this field study was to analyse the association among results of a serum enzyme-linked immunosorbent assays (ELISA), faecal culture and nested PCR tests on milk, blood and faeces for Mycobacterium avium subsp. paratuberculosis detection in dairy cows. Faeces, blood and milk samples were collected from 328 lactating dairy cows in four known infected herds. Results were analysed to determine associations and levels of agreement between pairs of tests. A total of 61 animals (18.6%) tested positive when all the tests were interpreted in parallel. The agreement between results in different pairs of tests was poor, slight and fair in two, five and three of the 10 possible combinations respectively. Faecal culture and faecal polymerase chain reaction (PCR) resulted in the highest kappa coefficient (0.39; fair agreement), with the lowest agreement being for ELISA and blood PCR (-0.036; poor agreement). Fisher's exact test resulted in statistically significant associations (P < or = 0.05) between the following test pairs: ELISA : faecal culture; ELISA : faecal PCR; milk PCR : faecal PCR, blood PCR : faecal PCR and faecal culture : faecal PCR. Enzyme-linked immunosorbent assays showed the highest complementary sensitivity values for all the possible two-test combinations, followed by faecal PCR. The combined use of ELISA and faecal PCR has the potential to increase the overall sensitivity for the diagnosis of paratuberculosis infection.