Heme levels and growth of malignant murine erythroleukemia cells in heme-free medium are drastically reduced by incubation of these cells in the presence of 4,6-dioxoheptanoic acid [succinylacetone (SA)]. When hematin was added to the culture medium of heme-depleted cells, the intracellular heme levels returned to normal, and growth inhibition produced by SA was also reversed. However, when cells depleted of heme by growth in heme-free medium containing SA were placed in heme-free medium without SA, heme levels were restored to normal, and growth was resumed. Hematin uptake in both untreated and heme-depleted malignant murine erythroleukemia cells exhibited biphasic kinetics, with a rapid phase of about 2 min followed by a slower uptake. The rate of uptake of exogenous hematin was slightly greater at 37° than at 20°. Although supplementation of heme-free medium with exogenous hematin increased total cellular heme in both untreated and heme-depleted malignant murine erythroleukemia cells, the fraction of heme in the 20,000 × g sediment was unaffected. A nonmalignant fibroblastic cell line, 3T3, exhibited little or no capacity to take up exogenous hematin.