Background Cocaine inhibition of dopamine (DA) uptake through the dopamine transporter (DAT) has been proposed to occur by both competitive and non-competitive mechanisms. Current pharmacological and computational data support the existence of two independent cocaine binding sites on DAT, a centrally located site (S1) and a more extracellularly positioned site (S2), which may underlie the complexity of cocaine inhibition of DAT. Objective: Identify the binding site(s) of the high-affinity cocaine photo-affinity analogs RTI 82, MFZ 2 24, and JHC 2 48. Results: Computational docking of the analogs to DAT as well as biochemical peptide mapping analyses of crosslinked DAT complexes revealed independent sites of adduction of the 4ʹ-azido-3ʹ-iodophenylethyl ester moiety on each analog. Our computational and crosslinking data place the core tropane pharmacophore in the S1 binding pocket, which was verified biochemically using the substituted cysteine accessibility method. Cocaine and the analogs could protect S1, but not S2 residues from reacting with methanethiosulfonate reagents. Conclusion The tropane pharmacophore binds to the S1 site, which overlaps the putative DA binding site, supporting a competitive mechanism for cocaine inhibition of DA uptake. Grant Support: INBRE Program of the NCRR and NIDA-IRP (AHN), DA027845 (LKH & RAV), P20 RR017699 from the COBRE Program of the NCRR, and P20 RR016741 from ND EPSCoR IIG (RAV & JDF).