Technical Abstract: Fumonisin B1 (FB1) is a fungal toxin found in maize worldwide. It is hepato- and nephrotoxic in many species, a liver and kidney carcinogen in rodents, and suspected to be involved in human disease. Its mechanism of action is disruption of sphingolipid metabolism consequent to inhibition of ceramide synthases (CerS). In cultured cells, inhibition of CerS by FB1 increases sphinganine (Sa), Sa 1-phosphate and a previously unidentified metabolite, believed to be a novel sphingoid base. In this study, the metabolite was analysed by mass spectrometry (MS) and assigned a m/z =286.3123 in positive ionization mode, consistent with a 1- or 3-deoxysphinganine (deoxySa), C18H40NO. Based on the calculated formula, it was hypothesized that the putative deoxySa was either produced from Sa via a dehydroxylase or was formed from the utilization of alanine by serine palmitoyl transferase (SPT), the first and rate-limiting enzyme in sphingolipid biosynthesis. Comparison with a synthetic standard using liquid chromatography tandem MS (LC-MS/MS) identified the metabolite as 1-deoxySa based on LC mobility and production of a distinctive m/z 44 fragment ion (CH3CH=NH2+) upon collision-induced dissociation. By comparison, Sa produced a distinctive m/z 60 fragment ion (HOCH2CH=NH2+). Inhibition of SPT with myriocin blocked the formation of both Sa and 1-deoxySA. Labeling studies using L-alanine-U-13C3 and L-serine-U-13C3 showed the preferential incorporation of alanine into 1-deoxySa, confirming that 1-deoxySA arises from condensation of alanine with palmitoyl-CoA via SPT.