In vitro covalent binding studies in which xenobiotics are shown to undergo metabolism-dependent covalent binding to macromolecules have been commonly used to shed light on the biochemical mechanisms of xenobiotic-induced toxicity. In this paper, 18 drugs (nine hepatotoxins and nine nonhepatotoxins) were tested for their proclivity to demonstrate metabolism-dependent covalent binding to macromolecules in human liver S-9 fraction (9000 g supernatant) or human hepatocytes, as an extension to previous work that used human liver microsomes published in this journal [ Obach et al. ( 2008 ) Chem. Res. Toxicol. 21 , 1814 -1822 ]. In the S-9 fraction, seven out of the nine drugs in each category demonstrated some level of metabolism-dependent covalent binding. Inclusion of reduced glutathione, cofactors needed by conjugating enzymes, and other parameters (total daily dose and fraction of total intrinsic clearance comprised by covalent binding) improved the ability of the system to separate hepatotoxins from nonhepatotoxins to a limited extent. Covalent binding in human hepatocytes showed that six out of the nine hepatotoxins and four out of eight nonhepatotoxins demonstrated covalent binding. Taking into account estimates of total daily body burden of covalent binding from the hepatocyte data showed an improvement over other in vitro systems for distinguishing hepatotoxins from nonhepatotoxins; however, this metabolism system still displayed some false positives. Combined with the previous study using liver microsomes, these findings identify the limitations of in vitro covalent binding data for prospective prediction of hepatotoxicity for new drug candidates and highlight the need for a better understanding of the link between drug bioactivation, covalent adduct formation, and toxicity outcomes. Directly relating covalent binding to hepatotoxicity is likely an oversimplification of the process whereby adduct formation ultimately leads to toxicity. Understanding underlying complexities (e.g., which macromolecules are important covalent binding targets, interindividual differences in susceptibility, etc.) will be essential to any understanding of the problem of metabolism-dependent hepatotoxicity and predicting toxicity from in vitro experiments.