A peptide contained in the venom of the predatory marine snail Conus tulipa, ρ-TIA, has previously been shown to possess α 1,-adrenoreceptor antagonist activity. Here, we further characterize its pharmacological activity as well as its structure-activity relationships. In the isolated rat vas deferens, ρ-TIA inhibited α1, -adrenoreceptor-mediated increases in cytosolic Ca2+ concentration that were triggered by norepinephrine, but did not affect presynaptic α2-adrenoreceptor-mediated responses. In radioligand binding assays using [125I]HEAT, ρ-TIA displayed slightly greater potency at the α1B, than at the α1A or α1D, subtypes. Moreover, although it did not affect the rate of association for [3H]prazosin binding to the α 1B-adrenoreceptor, the dissociation rate was increased, indicating non-competitive antagonism by ρ-TIA. N-terminally truncated analogs of ρ-TIA were less active than the full-length peptide, with a large decline in activity observed upon removal of the fourth residue of ρ-TIA (Arg 4). An alanine walk of ρ-TIA confirmed the importance of Arg 4 for activity and revealed a number of other residues clustered around Arg4 that contribute to the potency of ρ-TIA. The unique allosteric antagonism of ρ-TIA resulting from its interaction with receptor residues that constitute a binding site that is distinct from that of the classical competitive α1,-adrenoreceptor antagonists may allow the development of inhibitors that are highly subtype selective.