Cylindrospermopsin (CYN) is a toxic alkaloid-like compound produced by some strains of cyanobacteria, procariotic organisms occurring in water blooms, observed worldwide in eutrophic lakes and drinking water reservoirs. Methods for determination of CYN in freshwater and fish muscle by liquid chromatography coupled to electrospray ion trap mass spectrometry are herein described. The performances of both methods are reported; ion trap LC/ESI-MS/MS resulted highly selective and reliable in unambiguous identification of CYN, based on monitoring the precursor ion and three product ions. The methods developed showed satisfactory mean recoveries (higher than 63.6%) and relative standard deviations, ranging from 5.8 to 9.8%. The limits of quantification at 0.10 ng/mL in freshwaters and 1.0 ng/g in fish muscle, respectively, allow for determination of CYN also in early contamination stages. Ion trap LC/ESI-MS/MS was successfully applied to the identification and quantification of CYN in water and cyanobacteria extracts from Lake Averno, near Naples, representing the first case of contamination described in southern Italy. Copyright © 2009 John Wiley & Sons, Ltd.