To explore aqueous accessibility and functional contributions of transmembrane domain (TM) 1 in human serotonin transporter (hSERT) proteins, we utilized the largely methanethiosulfonate (MTS) insensitive hSERT C109A mutant and mutated individual residues of hSERT TM1 to Cys followed by tests of MTS inactivation of 5-hydroxytryptamine (5-HT) transport. Residues in TM1 cytoplasmic to Gly-94 were largely unaffected by Cys substitution, whereas the mutation of residues extracellular to Ile-93 variably diminished transport activity. TM1 Cys substitutions displayed differential sensitivity to MTS reagents, with residues more cytoplasmic to Asp-98 being largely insensitive to MTS inactivation. Aminoethylmethanethiosulfonate (MTSEA), [2-(trimethylammonium) ethyl]methanethiosulfonate bromide (MTSET), and sodium (2-sulfonatoethyl)-methanethiosulfonate (MTSES) similarly and profoundly inactivated 5-HT transport by SERT mutants D98C, G100C, W103C, and Y107C. MTSEA uniquely inactivated transport activity of S91C, G94C, Y95C but increased activity at I108C. MTSEA and MTSET, but not MTSES, inactivated transport function at N101C. Notably, 5-HT provided partial to complete protection from MTSET inactivation for D98C, G100C, N101C, and Y107C. Equivalent blockade of MTSET inactivation at N101C was observed with 5-HT at both room temperature and at 4 degrees C, inconsistent with major conformational changes leading to protection. Notably, cocaine also protected MTSET inactivation of G100C and N101C, although MTS incubations with N101C that eliminate 5-HT transport do not preclude cocaine analog binding nor its inhibition by 5-HT. 5-HT modestly enhanced the inactivation by MTSET at I93C and Y95C, whereas cocaine significantly enhanced MTSET sensitivity at Y107C and I108C. In summary, our studies reveal physical differences in TM1 accessibility to externally applied MTS reagents and reveal sites supporting substrate and antagonist modulation of MTS inactivation. Moreover, we identify a limit to accessibility for membrane-impermeant MTS reagents that may reflect aspects of an occluded permeation pathway.