To study the mechanism by which heme regulates the heme-regulated eIF-2 alpha kinase (HRI), the effects of various protoporphyrin IX (PP) compounds on the kinase activities and intersubunit disulfide formation of HRI and on protein synthesis in reticulocyte lysates were examined. Hemin and cobalt protoporphyrin (CoPP) are more effective than ZnPP, NiPP, SnPP, and metal-free PP in promoting intersubunit disulfide bond formation in HRI, in inhibiting the autokinase and eIF-2 alpha kinase activities of HRI, in inhibiting phosphorylation of eIF-2 alpha in rabbit reticulocytes, in maintaining protein synthesis, and in reversing the inhibition of protein synthesis in heme deficiency. There is an apparent correlation of in vitro intersubunit disulfide formation of HRI and the regulation of HRI kinase activities and protein synthesis by these porphyrin compounds. HRI in the reticulocyte lysate can be cross-linked by 1,6-bismaleimidohexane (bis-NEM). The formation of bis-NEM cross-linked dimers in lysates is prevented completely by N-ethylmaleimide (NEM) which alkylates free sulfhydryl groups and is diminished by hemin and CoPP. These results support the view that HRI in hemin-supplemented lysates is in equilibrium between the noncovalently linked dimer and the disulfide-linked dimer. The molecular size of HRI in control, hemin-supplemented, or NEM-treated hemin-supplemented lysates is identical to that of purified HRI; activation of HRI and changes in its thiol status do not significantly affect its molecular size.