An LC/MS analysis with diagnostic screening for the detection of peptides with posttranslational modifications revealed the presence of novel sulfated peptides within the α-conotoxin molecular mass range in Conus anemone crude venom. A functional assay of the extract showed activity at several neuronal nicotinic acetylcholine receptors (nAChRs). Three sulfated α-conotoxins (AnIA, AnIB, and AnIC) were identified by LC/MS and assay-directed fractionation and sequenced after purification. The most active of these, α-AnIB, was further characterized and used to investigate the influence of posttranslational modifications on affinity. Synthetic AnIB exhibited subnanomolar potency at the rat α3β2 nAChR (IC 50 0.3 nM) and was 200-fold less active on the rat α7 nAChR (IC50 76 nM). The unsulfated peptide [Tyr16]AnIB showed a 2-fold and 10-fold decrease in activities at α3β2 (IC50 0.6 nM) and α7 (IC50 836 nM) nAChR, respectively. Likewise, removal of the C-terminal amide had a greater influence on potency at the α7 (IC50 367 nM) than at the α3β2 nAChR (IC 50 0.5 nM). Stepwise removal of two N-terminal glycine residues revealed that these residues affect the binding kinetics of the peptide. Comparison with similar 4/7-α-conotoxin sequences suggests that residue 11 (alanine or glycine) and residue 14 (glutamine) constitute important determinants for α3β2 selectivity, whereas the C-terminal amidation and sulfation at tyrosine-16 favor α7 affinity.