Stereoselectivity is important in many pharmacological processes but its impact on drug membrane transport is scarcely understood. Recent studies showed strong stereoselective effects in the cellular uptake of fenoterol by the organic cation transporters OCT1 and OCT2. To provide possible molecular explanations, homology models were developed and the putative interactions between fenoterol enantiomers and key residues explored in silico through computational docking, molecular dynamics simulations, and binding free energy calculations as well as in vitro by site-directed mutagenesis and cellular uptake assays. Our results suggest that the observed 1.9-fold higher maximum transport velocity (v(max)) for (R,R)- over (S,S)-fenoterol in OCT1 is because the enantiomers bind to two distinct binding sites. Mutating PHE355 and ILE442, predicted to interact with (R,R)-fenoterol, reduced the v(max) ratio to 1.5 and 1.3, respectively, and to 1.2 in combination. Mutating THR272, predicted to interact with (S,S)-fenoterol, slightly increased stereoselectivity (vmax ratio of 2.2), while F244A resulted in a 35-fold increase in v(max) and a lower affinity (29-fold higher K-m) for (S,S)-fenoterol. Both enantiomers of salbutamol, for which almost no stereoselectivity was observed, were predicted to occupy the same binding pocket as (R,R)-fenoterol. Unlike for OCT1, both fenoterol enantiomers bind in the same region in OCT2 but in different conformations. Mutating THR246, predicted to interact with (S,S)-fenoterol in OCT2, led to an 11-fold decreased v(max). Altogether, our mutagenesis results correlate relatively well with our computational predictions and thereby provide an experimentally-corroborated hypothesis for the strong and contrasting enantiopreference in fenoterol uptake by OCT1 and OCT2.