Succinylacetone (SA, 4,6-dioxoheptanoic acid) inhibits d-aminolevulinic acid dehydrase, the second enzyme of the heme biosynthetic pathway and thereby inhibits heme biosynthesis. In the present study SA is shown to inhibit the growth of the Walker carcinosarcoma (W256) in vitro and in vivo, the Novikoff hepatoma in vivo, and L1210 leukemia in vitro, but only slightly in vivo. Rats can tolerate significantly larger doses of SA for at least twice as long as were administered in the present study without gross evidence of toxicity. In contrast to findings in previously published studies with murine erythroleukemia cells, the inhibition of growth of L1210 and W256 cells by SA in vitro is not accompanied by a decrease in cellular heme and is not reversed by addition of hematin to the medium. This suggests a second mechanism of growth inhibition of tumor cells unrelated to heme biosynthesis. Although the growth of both W256 and L1210 cells was markedly inhibited by the same concentration of SA in culture, there was a great difference in responsiveness in vivo, in that much greater inhibition of the growth of the Walker tumor was produced by SA.