Acylfulvenes (AFs), a class of semisynthetic analogues of the sesquiterpene natural product illudin S, are cytotoxic toward cancer cells. The minor structural changes between illudin S and AFs translate to an improved therapeutic window in preclinical cell-based assays and xenograft models. AFs are, therefore, unique tools for addressing the chemical and biochemical basis of cytotoxic selectivity. AFs elicit cytotoxic responses by alkylation of biological targets, including DNA. While AFs are capable of direct alkylation, cytosolic reductive bioactivation to an electrophilic intermediate is correlated with enhanced cytotoxicity. Data obtained in this study illustrate chemical aspects of the process of AF activation. By tracking reaction mechanisms with stable isotope-labeled reagents, enzymatic versus chemical activation pathways for AF were compared for reactions involving the NADPH-dependent enzyme prostaglandin reductase 1 (PTGR1) or sodium borohydride, respectively. These two processes resulted in isomeric products that appear to give rise to similar patterns of DNA modification. The chemically activated isomer has been newly isolated and chemically characterized in this study, including an assessment of its relative stereochemistry and stability at varying pH and under bioassay conditions. In mammalian cancer cells, this chemically activated analogue was shown to not rely on further cellular activation to significantly enhance cytotoxic potency, in contrast to the requirements of AF. On the basis of this study, we anticipate that the chemically activated form of AF will serve as a useful chemical probe for evaluating biomolecular interactions independent of enzyme-mediated activation.