Chicken erythrocytes, which contain less DNA than mammalian diploid cells, were used as an internal standard to control instrumental and staining variables during flow microfluorometric analysis. With the DNA stain, mithramycin, and with an EPICS II flow microfluorometer, ratios between the modal G1 fluorescence of experimental cells and that of chicken erythrocytes were determined. The results indicate that unperturbed cell populations of L1210 mouse leukemia and HeLa human cervical cancer cells in vitro and L1210 ascites cells in vivo have relatively stable fluorescence ratios, although there is a significant difference between the ratios of 1 L1210 cell line in vitro and another in vivo. L1210 ascites treated in vivo with different schedules of cyclophosphamide and adriamycin showed wide fluctuations in the fluorescence intensity ratios for 96 h after treatment. Differences in the fluorescence ratios were observed between less advanced and more advanced L1210 ascites after treatment with the same schedule. These effects indicate an alteration in DNA staining with mithramycin, brought about by drug treatment that could seriously affect the interpretation of DNA histogram data. Changes in mithramycin staining may be an important probe to detect persistent drug effects.