Flow cytometry was used to determine the daunomycin content of rat bone marrow cells after incubation in vitro. The spontaneous fluorescence of daunomycin was measured upon excitation with laser light at 488 nm. Forward and perpendicular light scatters of the cells were simultaneously measured to allow identification of granulocytic and lymphocytic subpopulations. A linear relationship was found for a 30 min exposure between the drug concentration (ranging from 0.2 to 3 .mu.g/ml) in the incubation medium and the fluorescence intensity for lymphocytes and granulocytes. Dead cells contaminating cell suspensions showed several times higher daunomycin fluorescence than did viable cells. In fixed cells, the fluorescence reflects daunomycin bound to DNA, since the fluorescence intensity of daunomycin-treated fixed cells returns to the level of unstained cells after DNase treatment. Quantitation of the cellular drug concentration was done by exposing granulocytes and lymphocytes in vitro to varying doses of [3H]daunomycin. The concentration per cell was on the order of 10-18 mol. For both the granulocytic and the lymphocytic subpopulations, a linear relationship was found between drug-related radioactivity and fluorescence intensity. [Daunomycin is an antineoplastic drug.].