Sequence detection by the 5' nuclease TaqMan assay uses online detection of internal fluorogenic probes in closed PCR tubes. Primers and probe were chosen from a part of the omlA gene common to all serotypes of Actinobacillus pleuropneumoniae, which gave an amplicon of 92 bp. The test was evaluated with 73 lung isolates and 120 tonsil isolates of A. pleuropneumoniae as well as with a collection of reference strains. By using a C_t value (cycle number in which the fluorescence exceeds the threshold defined by the software) of 30 as the cutoff limit, the 5' nuclease assay represents a test with 100% sensitivity and 100% specificity. A high degree of reproducibility of the test was demonstrated. If samples with C_t values of 30 are considered positive, the detection limit of the assay was 1 CFU/reaction tube, corresponding to a 10-fold higher number of DNA templates. After cycle 30, nonspecific reactions appeared when testing dilutions of DNA templates or pure cultures of A. pleuropneumoniae, as well as when testing tonsil scrapings from specific-pathogen-free herds. The diagnostic sensitivity, as evaluated with 586 tonsil scrapings from animals infected with A. pleuropneumoniae, is the same level as that of a PCR test based on the omlA gene described previously. The 5' nuclease assay represents a fast method for species-specific detection and identification of A. pleuropneumoniae in pure and mixed cultures. The evaluation shows, however, that a C_t value cutoff limit of 30 must be chosen in order to obtain reliable results. The investigation emphasizes that a thorough evaluation of the criteria used to define a positive test result is necessary.