AbstractSynthetic cathinones, one of the most prevalent categories of new psychoactive substances, have been posing a serious threat to public health. Methylmethcathinones (MMCs), notably 3-MMC, have seen an alarming increase in their use in the last decade. The metabolism and toxicology of a large majority of synthetic cathinones, including 3-MMC and 2-MMC, remain unknown. Traditionally, male-derived liver materials have been used as in vitro metabolic incubations to investigate the metabolism of xenobiotics, including MMCs. Therefore, little is known about the metabolism in female-derived in vitro models and the potential sex-specific differences in biotransformation. In this study, the metabolism of 2-MMC, 3-MMC, and 4-MMC was investigated using female rat and human liver microsomal incubations, as well as male rat and human liver microsomal incubations. A total of 25 phase I metabolites of MMCs were detected and tentatively identified using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Seven sex-specific metabolites were detected exclusively using pooled male rat liver microsomal incubations. In addition, the metabolites generated from the sex-dependent in vitro metabolic incubations that were present in both male and female rat liver microsomal incubations showed differences in relative abundance. Yet, neither sex-specific metabolites nor significant differences in relative abundance were observed from pooled human liver microsomal incubations. This is the first study to report the phase I metabolic pathways of MMCs using in vitro metabolic incubations for both male and female liver microsomes, and the relative abundance of the metabolites observed from each sex. Graphical abstract