Mutation of a conserved Asp (D98) in the rat serotonin (5HT) transporter (rSERT) to Glu (D98E) led to decreased 5HT transport capacity, diminished coupling to extracellular Na+ and Cl-, and a selective loss of antagonist potencies (cocaine, imipramine, and citalopram but not paroxetine or mazindol) with no change in 5HT Km value. D98E, which extends the acidic side chain by one carbon, affected the rank-order potency of substrate analogs for inhibition of 5HT transport, selectively increasing the potency of two analogs with shorter alkylamine side chains, gramine, and dihydroxybenzylamine. D98E also increased the efficacy of gramine relative to 5HT for inducing substrate-activated currents in Xenopus laevis oocytes, but these currents were noticeably dependent on extracellular medium acidification. I-V profiles for substrate-independent and -dependent currents indicated that the mutation selectively impacts ion permeation coupled to 5HT occupancy. The ability of the D98E mutant to modulate selective aspects of substrate recognition, to perturb ion dependence as well as modify substrate-induced currents, suggests that transmembrane domain I plays a critical role in defining the permeation pathway of biogenic amine transporters.