A newly developed flow cytometry technique for simultaneous measurements of three features of individual cells--DNA, RNA, and nuclear diameter--using acridine orange as a fluorescent metachromatic dye, has been applied to cell-cycle analysis. DNA stemline determination, and to classification of 102 cases of human leukemias in adults. Acute lymphoblastic leukemia (L1-2) was characterized by moderately increased RNA of G0/1 cells as compared to normal lymphocytes; acute nonlymphoblastic leukemia (M 1-5) by very high RNA of G0/G1 cells. Both had either diploid or aneuploid DNA stemlines. Chronic lymphocytic leukemia showed diploid DNA, very low proliferation, and low RNA, similar to that found by use to be typical for normal B cells. In chronic myelogenous leukemia, two cell populations were distinguished, one with high RNA, the other with very low RNA and elongated nuclear diameter due to stripped, unfolded nuclei of polymorphonuclear leukocytes. The number of leukemic blast cells, identified by aneuploid DNA values, correlates well with conventional microscopy counts and could be followed during the course of treatment. Thus, acridine orange flow cytometry can be used to discriminate subtypes of human leukemias, to determine cell cycle stages, and to detect and monitor aneuploid leukemia stemlines.