Cone snail venom is a complex mixture of cysteine-rich peptides, many of which have interesting bioactivities and are useful as pharmacological tools. There are different pharmacological families of cone snail venom peptides, also known as conotoxins, that can have differing affects. CIA is a member of the a-conotoxin family and was originally isolated from the cone snail Conus catus. Native CIA is a typical 3/5 α-conotoxin that potently blocks the mammalian muscle type nAChR (IC50 5.7 nM) but also the neuronal rat nAChR subtype α3β2 (IC50 2.06 µM). We have synthesized three cyclic analogues of α-conotoxin CIA and determined their three-dimensional structures based on NMR spectroscopy data. The peptide containing the two amino acid linker has a slightly different structure to the other peptides, and this change appears to be influencing the bioactivity. For example, in vivo injections into adult zebrafish revealed that all peptides were highly paralytic to fish, the natural prey of C. catus, with native CIA and its two amino acid linker analogue being the most potent. Interestingly, the peptide containing a three amino acid linker has ~500-fold greater potency at the α3β2 neuronal nAChR subtype compared to the native conotoxin, whereas the activity at the muscle nAChR was similar for all peptides. We show that both native CIA and the cyclic analogue containing a two amino acid linker are capable of immobilizing zebrafish larvae when incubated in water.