The determinants of cellular net uptake of three anthracyclines—daunorubicin, adriamycin, and rubidazone—were studied in Ehrlich ascites tumour cells in vitro. A striking finding was the marked effect of the pH on net uptake in the way that lowering of the pH decreased the rate of uptake and steady-state level. As p K A of the drugs proved to be 8.15–8.45, this finding suggests a penetration of the drugs by passive transport of the unionized form of the molecules. Several findings indicated an uphill efflux of the drugs from the cells. (1) The estimate of the cytoplasm/medium concentration ratio at steady-state for daunorubicin and rubidazone was considerably below that predicted when assuming passive distribution. (2) The cellular accumulation of the drugs was significantly enhanced by sodium azide. (3) The cellular release of the drugs was inhibited by sodium azide. Together these findings suggest a membrane transport of the drugs as a “leak-and-pump” system. Studies of the intracellular binding showed that the affinity of adriamycin for isolated nuclei and for total cellular homogenate was about 50 per cent higher than that of daunorubicin; rubidazone possessed the lowest affinity for cellular binding sites. The affinity of the drugs for intracellular binding sites was not influenced by the pH. The results indicate that the net uptake of anthracycline derivatives in these tumour cells is determined by: (1) the permeability of the membrane to the unionized form of the molecule ; (2) the specificity of the drug for the mechanism of active efflux; (3) the relationship between intracellular and extracellular pH; (4) the p K A, value of the drug; (5) the affinity of the drug for the intracellular binding sites.