Aims: The potential of using flow cytometry (FC) in combination with a fluorescent dye (SYBR green-I) for rapidly estimating Mycoplasma mycoides subSPS. mycoides large-colony type (MmmLC) in broth culture was examined. Methods and Results: The FC analysis was performed by staining the MmmLC cells with a fluorescent dye, SYBR green-I (SYBR), and the results were compared with plate count method (colony forming units, -CFUs). There was a good correlation (linear regression, r(2) = 0.93) between mycoplasma counts determined by FC (cells ml(-1)) and by traditional plate count method (CFU ml(-1)). The lowest bacterial concentration detected by FC and traditional plate count was of the order of 10(4) cells ml(-1) and 10(3) CFU ml(-1), respectively. FC method allowed results in 20-30 min, whereas at least 24 h were necessary to obtain results with the traditional plate count method (CFU). Conclusion: Growth rates of MmmLC in broth medium determined by FC were highly reproducible and correlated well with mycoplasma counts assessed by the plate count method. Significance and Impact of the Study: These findings suggest that FC could be a good alternative to replace other time-consuming techniques that are currently used to enumerate mycoplasma in broth medium, such as plate count method (CFU).