α-Conotoxins from Conus snails are indispensable tools for distinguishing various subtypes of nicotinic acetylcholine receptors (nAChRs), and synthesis of α-conotoxin analogs may yield novel antagonists of higher potency and selectivity. We incorporated additional positive charges into α-conotoxins and analyzed their binding to nAChRs. Introduction of Arg or Lys residues instead of Ser12 in α-conotoxins GI and SI, or D12K substitution in α-conotoxin SIA increased the affinity for both the high- and low-affinity sites in membrane-bound Torpedo californica nAChR. The effect was most pronounced for [D12K]SIA with 30- and 200-fold enhancement for the respective sites, resulting in the most potent α-conotoxin blocker of the Torpedo nAChR among those tested. Similarly, D14K substitution in α-conotoxin [A10L]PnIA, a blocker of neuronal α7 nAChR, was previously shown to increase the affinity for this receptor and endowed [A10L,D14K]PnIA with the capacity to distinguish between acetylcholine-binding proteins from the mollusks Lymnaea stagnalis and Aplysia californica. We found that [A10L,D14K]PnIA also distinguishes two α7-like anion-selective nAChR subtypes present on identified neurons of L. stagnalis: [D14K] mutation affected only slightly the potency of [A10L]PnIA to block nAChRs on neurons with low sensitivity to α-conotoxin ImI, but gave a 50-fold enhancement of blocking activity in cells with high sensitivity to ImI. Therefore, the introduction of an additional positive charge in the C-terminus of α-conotoxins targeting some muscle or neuronal nAChRs made them more discriminative towards the respective nAChR subtypes. In the case of muscle-type α-conotoxin [D12K]SIA, the contribution of the Lys12 positive charge to enhanced affinity towards Torpedo nAChR was rationalized with the aid of computer modeling. © 2006 The Authors.