The correlation between intracellular accumulation of doxorubicin (DOX) and drug cytotoxicity was studied in cells from seven cell lines of rodent and human origin. We used three Chinese hamster ovary cell lines (AuxB1, CH^RC5, and UV20), two murine tumor cell lines (KHT-LP1 and EMT6/Ro), and two human tumor cell lines (MGH-U1 and DLD-1). Intracellular DOX was measured by its fluorescence intensity with flow cytometry, and drug cytotoxicity was quantified with clonogenic assays. When data for all of the cell lines were combined, cell killing was correlated with the intracellular concentration of DOX (r = -.88). For the cell line AuxB1 and its DOX-resistant subline CH^RC5, in which cells express high levels of P-glycoprotein, the relationship between cell survival and the intracellular concentration of DOX was stronger than that for the other cell lines. These results suggest that differences in intracellular accumulation of DOX account for much of the heterogeneity in response to the drug in cells from different cell lines, although additional mechanisms also contribute to variation in drug sensitivity. Flow cytometric analysis of intracellular DOX fluorescence is a simple assay that should be tested in cells from human tumors as a possible predictor of tumor response. For a given cell line, this technique also provides a rapid way to monitor the development of drug resistance after multiple courses of chemotherapy. [J Natl Cancer Inst 1989;81:55–59]