Aims: In the present study we evaluated the potential application of the flow cytometry (FC) technique to determine the growth rates of Mycoplasma hyopneumoniae in a broth medium. Methods and Results: The FC analysis was performed using the fluorochromes Syto 9, propidium iodide (PI) or a combination of both dyes and results were compared with those obtained by colour-changing units (CCU) and pH measurements. While CCU counts ml(-1) were higher than those obtained from the FC technique, a good relation between M. hyopneumoniae growth rates was observed in the different phases of the growth curve ( logarithmic, stationary and senescence phases). Labelling with Syto 9 alone was sufficient to differentiate M. hyopneumoniae cells with different amounts of nucleic acids, in the stationary and senescence phase of the M. hyopneumoniae growth curve. PI labelling did not detect cell death in the end phase of M. hyopneumoniae growth. Conclusions: These data show that FC is a very useful, practical and fast technique to study the growth rates of M. hyopneumoniae in broth medium. Significance and Impact of the Study: This is to our knowledge the first application of FC to the study of M. hyopneumoniae growth in broth culture.