A series of ortho-substituted bromobenzene derivatives with widely differing hepatotoxicities were investigated for their tendency to undergo oxidative metabolism and protein covalent binding in the presence of rat liver microsomes in vitro. Compounds studied included o-bromobenzonitrile, o-dibromobenzene, bromobenzene, o-bromoanisole, and o-bromotoluene (names in order of decreasing hepatotoxicity and increasing rate of in vitro metabolism). No correlation was found between net covalent binding and toxicity. However, a good rank order correlation was observed between toxicity and the relative binding index of each compounds, defined as (picomoles covalently bound/nmol metabolized), with values ranging from a low of 7 with o-bromotoluene to a high of 365 with o-bromobenzonitrile, Extensive side chain metabolism was observed with o-bromotoluene (92-95%) and o-bromoanisole (26-42%), which accounts for their high rate of total metabolism and low relative binding index. The extent of tritium loss, relative to C-14, was assessed for each compound as an index of the "average oxidation state" of those metabolites, presumably epoxides and/or quinones, which covalently bound to protein. Tritium retention ranged from a high of 84% with o-bromobenzonitrile to a low of 21% with o-bromoanisole, and generally paralleled toxicity. These results show that introduction of ortho-substituents onto bromobenzene leads to qualitative and quantitative changes in overall oxidative metabolism, as well as important qualitative changes in the nature of reactive metabolites formed. Nevertheless, the relative binding index computed for each compound appears to reconcile these changes to a large degree, giving an in vitro index which correlates well with toxicity.