We developed a fluorometric system which does for broth-grown mycoplasmas what turbidimetric analysis does for broth-grown bacteria. It allows one to monitor the growth of broth-grown mycoplasmas at any interval desired. The entire procedure is quick, taking not more than 20 min. The fluorometric readings correlate with colonial growth on agar, making it possible, for the first time, to take readings which closely estimate the CFU present in the culture at a given moment in time. We show that this system can be used to assess the effectiveness of an antimycoplasmal antibiotic and to optimize medium components and that fluorometer readings taken during the logarithmic phase of growth correlate with the DNA content of the viable cells. Use of this methodology will permit investigators to know absolutely the phase of the growth cycle of the culture concomitant with the growth of the culture itself, and since fluorometer readings of culture aliquots can be converted to DNA equivalents, the standardization of mycoplasmal cultures within and between laboratories will be a possibility.