The novel nicotinic receptor (nAChR) antagonist, N,N'-dodecane-1,12-diyl-bis-3-picolinium dibromide (bPiDDB), and its chemically reduced analog, r-bPiDDB, potently inhibit nicotine-evoked dopamine (DA) release from rat striatal slices. Since tobacco smokers self-administer nicotine repeatedly, animal models incorporating repeated nicotine treatment allow for mechanistic evaluation of therapeutic candidates following neuroadaptive changes. The current study determined the ability of bPiDDB, r-bPiDDB and alpha-conotoxin MII (alpha-CtxMII), a peptide antagonist selective for alpha6beta2-containing nAChRs, to inhibit nicotine-evoked [(3)H]DA release from striatal slices from rats repeatedly administered nicotine (0.4mg/kg for 10 days) or saline (control). Concomitant exposure to maximally effective concentrations of r-bPiDDB (1nM) and alpha-CtxMII (1nM) resulted in inhibition of nicotine-evoked [(3)H]DA release no greater than that produced by either antagonist alone, suggesting that r-bPiDDB inhibits alpha6beta2-containing nAChRs. Repeated nicotine treatment increased locomotor activity, demonstrating behavioral sensitization. Concentration-response curves for nicotine-evoked [(3)H]DA release were not different between nicotine-treated and control groups. Maximal inhibition for alpha-CtxMII was greater following repeated nicotine compared to control (I(max)=90% vs. 62%), with no change in potency. bPiDDB was 3-orders of magnitude more potent in inhibiting nicotine-evoked [(3)H]DA release in nicotine-treated rats compared to control rats (IC(50)=5pM vs. 6nM), with no change in maximal inhibition. Neither a shift to the left in the concentration response nor a change in maximal inhibition was observed for r-bPiDDB following repeated nicotine. Thus, repeated nicotine treatment may differentially regulate the stoichiometry, conformation and/or composition of alpha6beta2-containing nAChRs mediating nicotine-evoked striatal DA release. Therefore, bPiDDB and r-bPiDDB appear to target different alpha6beta2-containing nAChR subtypes.