Nicotinic acetylcholine receptors (nAChRs) are involved in the regulation of intracellular Ca2+-dependent processes both in normal and pathological states. α-Conotoxins from the venom of Conus marine mollusks are a valuable tool for the investigation of the pharmacological action and functional role of nAChRs. Analogues of α-conotoxin MII labeled by Bolton-Hunter reagent (BH-MII) or fluorescein isothiocyanate (FITC-MII) on the N-terminal glycine residue have been synthesized in the present work. Fluorescence microscopy studies of SH-SY5Y neuroblastoma cells loaded with Ca2+indicator Fura-2, or by both Ca2+indicator Fluo-4 and Na+indicator SBFI, were used to test the effect of MII modification on its ability to block Ca2+and Na+signals induced by nicotine. Measurements in SH-SY5Y cells showed that kinetics of the increase and recovery of the concentration of free Ca2+([Ca2+]i) upon nicotine application and washout was different from that for free Na+([Na+]i), this being evidence of differences in the mechanism of Ca2+and Na+homeostasis regulation. MII suppressed the nicotine-induced increase of [Ca2+]iand [Na+]iin a concentration-dependent manner. An additional tyrosine residue added to the N-terminus of one of the MII derivatives caused a significant decrease in the inhibitory action of MII; this decrease was even more pronounced when a large FITC label was introduced into MII. The BH-MII derivative had an inhibitory effect similar to that of unmodified α-conotoxin. MII and its iodinated derivatives are promising tools for radioligand assays. © Pleiades Publishing, Ltd., 2012.