Site-directed alkylation of consecutively introduced cysteines was employed to probe the solvent-accessible profile of highly conserved transmembrane helix 3 (TM3), spanning residues V127-T149 of the apical sodium-dependent bile acid transporter (ASBT), a key membrane protein involved in cholesterol homeostasis. Sequence alignment of SLC10 family members has previously identified a signature motif (ALGMMPL) localized to TM3 of ASBT with as yet undetermined function. Cysteine mutagenesis of this motif resulted in severe decreases in uptake activity only for mutants M141C and P142C. Additional conservative and nonconservative replacement of P142 suggests its structural and functional importance during the ASBT transport cycle. Significant decreases in transport activity were also observed for three cysteine mutants clustered along the exofacial half of the helix (M129C, T130C, S133C) and five mutants consecutively lining the cytosolic half of TM3 (L145C-T149C). Measurable surface expression was detected for all TM3 mutants. Using physicochemically different alkylating reagents, sites predominantly lining the cytosolic half of the TM3 helix were found to be solvent accessible (i.e., S128C, L143C-T149C). Analysis of substrate kinetics for select TM3 mutants demonstrates significant loss of taurocholic acid affinity for mutants S128C and L145C-T149C. Overall, we conclude (i) the functional and structural importance of P142 during the transport cycle and (ii) the presence of a large hydrophilic cleft region lining the cytosolic half of TM3 that may form portions of the substrate exit route during permeation. Our studies provide unique insight into molecular mechanisms guiding the ASBT transport cycle with respect to substrate binding and translocation events.