Our previous work suggested that collapsing the Na(+) gradient and membrane potential converts the dopamine (DA) transporter (DAT) to an inward-facing conformation with a different substrate binding profile. Here, DAT expressing human embryonic kidney 293 cells were permeabilized with digitonin, disrupting ion/voltage gradients and allowing passage of DAT substrates. The potency of p-tyramine and other non-catechols (d-amphetamine, beta-phenethylamine, MPP(+)) in inhibiting cocaine analog binding to DAT in digitonin-treated cells was markedly weakened to a level similar to that observed in cell-free membranes. In contrast, the potency of DA and another catechol, norepinephrine, was not significantly changed by the same treatment, whereas epinephrine showed only a modest reduction. These findings suggest that catechol substrates interact symmetrically with both sides of DAT and non-catechol substrates, favoring binding to outward-facing transporter. In the cocaine analog binding assay, the mutant W84L displayed enhanced intrinsic binding affinity for substrates in interacting with both outward- and inward-facing states; D313N showed wild-type-like symmetric binding; but D267L and E428Q showed an apparent improvement in the permeation pathway from the external face towards the substrate site. Thus, the structure of both substrate and transporter play a role in the sidedness and mode of interaction between them.