System L is a heterodimeric Na+-independent amino acid (aa) membrane transporter that mediates the transport of essential aa’s. System L activity has been demonstrated in the microvillous (MVM) and basal (BM) plasma membranes of the human placental epithelial exchange barrier, the syncytiotrophoblast. System L transporters are thought to act as obligatory 1:1 aa exchangers, transporting one aa in exchange for another. However, other modes of carrier-mediated transport are implicated; system L substrates can be transported across MVM and BM under zero-trans conditions, without exchangeable aa. This led to our hypothesis that system L-mediated transport may not occur solely via an obligatory exchange mechanism. To investigate this, we applied an integrated mathematical modelling approach to predict how transport of serine (Ser), a system L substrate, would behave under theoretical models of transport. Mathematical predictive models of obligatory aa exchangers, along with facilitated transport, were developed based on assumptions of carrier-mediated transport. Exchanger transporters were assumed to bind aa to confer 1:1 transport across the plasma membrane, whilst facilitated transport was assumed to be driven by the transmembrane aa gradient. The model predicted that for an exchanger together with a facilitated (slower rate) transport component, an outwardly directed substrate gradient would lead to an overshoot of substrate accumulation above tracer equilibrium concentration (Fig 1A).To test the models, we measured uptake of 7.5µM 14C-Ser (tracer) into MVM vesicles isolated from normal term placentas, in vesicles preloaded with 250µM (equal to extravesicular Ser concentration) or 1mM Ser (outwardly directed substrate gradient). Uptake of 14C-Ser mediated by system L was taken to be that inhibitable by 20mM 2-amino-2-norbornanecarboxylic acid (BCH), a system L substrate. In the presence of equal intravesicular and extravesicular Ser (250µM), 14C-Ser accumulated in a time-dependent manner to reach tracer equilibrium concentration (7.5µM; assuming an intravesicular volume of 1 µl/mg protein) by 10min, indicating uptake of 14C-Ser in the absence of a transmembrane substrate gradient. However, an outwardly directed substrate concentration gradient evoked a rapid overshoot of 14C-Ser accumulation above tracer equilibrium concentration, which was abolished by BCH (Fig 1B). These observations suggest trans-stimulation of system L activity showed good correspondence to model predictions with both an exchanger and facilitated transport component, suggesting system L activity in MVM vesicles is mediated by both components, although their molecular identities remain to be determined. This study emphasizes the utility of mathematical modelling in predicting aa modes of transport, providing an improved quantitative understanding of placental aa transporter mechanisms.