Isolation of Mycobacterium bovis from suspected cases of bovine tuberculosis demands laborious and time-consuming procedures. Also, direct PCR procedures on tissue samples show poor sensitivity, whereas radiometric and fluorescence-based identification procedures demand high running costs and do not reduce the time needed for isolation to less than 10 to 15 d. Owing to the aforementioned obstacles, the human macrophage cell line THP-1 and other macrophage cell lines were investigated in experiments of M. bovis propagation and isolation from organ samples. Macrophage cells can support a high-titered propagation within 48 h of minute amounts of both BCG and fully pathogenic M. bovis strains from organ samples. A proper antibiotic mixture prevents contamination of cell cultures. A seminested PCR for tuberculosis complex-specific insertion sequence IS6110 revealed M. bovis infection in infected cells. The same result can be obtained by a flow cytometry assay for expression of M. bovis chaperonin 10. The reduced time for isolation and identification of M. bovis (48-72 h) and the consistency of the test results make the use of macrophage cell lines attractive and cost-effective for veterinary laboratories involved in surveillance of bovine tuberculosis.