Ethylene dibromide (EDB), a known stomach carcinogen, and ethylene dichloride (EDC), which is carcinogenic to the liver, have been shown in in vitro experiments to bind covalently to stomach and hepatic microsomal proteins and to salmon sperm DNA. The binding of EDB or EDC with proteins was not significant when denatured microsomes were used or when DNA was used in the absence of microsomes. The binding of EDB to these macromolecules was augmented with increasing concentrations of microsomes. SKF-525A, an inhibitor of the microsomal metabolism of various substrates, significantly inhibited the binding of EDB to protein and DNA. These findings suggest that metabolic activation of EDB and EDC is required for their covalent binding to macromolecules. Glutathione and 1-methyl-2-mercaptolmidazole markedly decreased the binding of EDB, which indicated that a reactive electrophilic intermediate(s) of EDB is (are) involved in the binding. The binding of EDC to liver proteins of (C57BL/6 X C3//He)F1 mice, which are susceptible to liver tumor induction by EDC and to DNA, was significantly higher than the corresponding binding for Osborne-Mendel rats, a species not susceptible to liver tumor induction by this compound.