We have investigated the effect of succinylacetone (4,6-dioxoheptanoic acid) on hemoglobin synthesis and iron metabolism in reticulocytes. Succinylacetone, 0.1 and 1 mM, inhibited [2-14C]glycine incorporation into heme by 91.2 and 96.4%, respectively, and into globin by 85 and 90.2%, respectively. 60 microMM hemin completely prevented the inhibition of globin synthesis by succinylacetone, indicating that succinylacetone inhibits specifically the synthesis of heme. Added porphobilinogen, but not delta-aminolevulinic acid, partly overcame the inhibition of 59Fe incorporation into heme caused by succinylacetone suggesting that the drug inhibits delta-aminolevulinic acid dehydratase in reticulocytes. Succinylacetone, 10 microM 0.1 and 1 mM, inhibited 59Fe incorporation into heme by 50, 90 and 93%, respectively, but stimulated reticulocyte 59Fe uptake by about 25-30%. In succinylacetone-treated cells 59Fe accumulates in a fraction containing plasma membranes and mitochondria as well as cytosol ferritin and an unidentified low molecular weight fraction obtained by Sephacryl S-200 chromatography. Reincubation of washed succinylacetone- and 59Fe-transferrin-pretreated reticulocytes results in the transfer of 59Fe from the particulate fraction (plasma membrane plus mitochondria) into hemoglobin and this process is considerably stimulated by added protoporphyrin. Although the nature of the iron accumulated in the membrane-mitochondria fraction in succinylacetone-treated cells is unknown some of it is utilizable for hemoglobin synthesis, while cytosolic ferritin iron would appear to be mostly unavailable for incorporation into heme.