A quantitative real-time polymerase chain reaction (PCR) procedure followed by melting curve analysis, using the green fluorescence dye SYBR Green I, was developed for rapid detection and differentiation of mycoplasma contaminants in cell cultures. This method showed that the detection of the target sequence was linear over a range from 10(4) to 10 colony-forming units (CFU) of the mycoplasma cells. Analysis of the melting temperature of the PCR products allowed differentiation of the major mycoplasma contaminants. These results demonstrate that the protocol described in the present study can decrease the time to obtain reproducible results by simultaneous detection and differentiation of the Mycoplasma species contaminating cell cultures.