-Conopeptide MrIA (-MrIA) is a thirteen residue peptide contained in the venom of the predatory marine snail Conus marmoreus that has been found to inhibit the norepinephrine transporter (NET). We investigated whether -MrIA targeted the other members of the monoamine transporter family and found no effect of the peptide (100 M) on the activity of the dopamine transporter and the serotonin transporter, indicating a high specificity of action. The binding of the NET inhibitors, [³H]-nisoxetine and [³H]-mazindol, to the expressed rat and human NET was inhibited by -MrIA, with the conopeptide displaying a slight preference toward the rat isoform. For both radioligands, saturation binding studies showed that the inhibition by -MrIA was competitive in nature. It has previously been demonstrated that -MrIA does not compete with norepinephrine, unlike classically described NET inhibitors, such as nisoxetine and mazindol, which do. This pattern of behaviour implies that the binding site for -MrIA on the NET overlaps the antidepressant binding site and is wholly distinct from the substrate binding site. The inhibitory effect of -MrIA was found to be dependent on Na⁺, with the conopeptide becoming a less effective blocker of [³H]-norepinephrine by the NET under conditions of reduced extracellular Na⁺. In this respect, -MrIA is similar to the antidepressant inhibitors of the NET. The structure-activity relationship of -MrIA was investigated by alanine scanning. Four residues in the first cysteine-bracketed loop of -MrIA, and a His in loop 2 played a dominant role in the interaction between MrIA and the NET. H chemical shift comparisions indicated that side-chain interactions at these key positions were structurally perturbed by replacement of Gly6. From this data we present a model of -MrIA that shows the relative orientation of the key binding residues. This model provides a new molecular caliper for probing the structure of the NET.