Aims: A triplex real-time PCR assay to quantify Mycoplasma hyopneumoniae in specimens from live and dead pigs was developed and validated. The minimal dose of Myc. hyopneumoniae required to induce pneumonia in specific pathogen-free pigs was determined. Methods and Results: This TaqMan test simultaneously detected three genes encoding the proteins P46, P97 and P102. All Myc. hyopneumoniae strains analysed were detected, including strains isolated in three countries (France, England and Switzerland) and from several pig farms (n = 33), and the test was specific. The estimated detection thresholds were 1 center dot 3 genome equivalents (mu l-1) for the targets defined in p97 and p102 genes and 13 genome equivalents (mu l-1) for the segment defined in the p46 gene. This test was used to quantify Myc. hyopneumoniae in specimens sampled from experimentally infected pigs. In live pigs, c. 107, 108 and 1010 genome equivalents (ml-1) of Myc. hyopneumoniae were detected in the nasal cavities, tonsils and trachea samples, respectively. In dead pigs, 108-1010 genome equivalents (ml-1) of Myc. hyopneumoniae were detected in the lung tissue with pneumonia. The estimated minimal dose of Myc. hyopneumoniae required to induce pneumonia was 105 colour-changing units (CCU) per pig (corresponding to 108 mycoplasmas). Conclusion: The triplex RT-PCR test was validated and can be used for testing samples taken on the pig farms. Significance and Impact of the Study: This test should be a very useful tool in pig herds to control enzootic pneumonia or healthy carrier pigs and to study the dynamics of Myc. hyopneumoniae infections.