Cyanobacteria are a small group of photosynthetic planktonic bacteria, producing a large group of strong hepatotoxins called microcystins (MCs). Many studies have been conducted to evaluate the presence of MCs and nodularin (NOD) in water or in marine organisms, but little research has been done on the atmospheric environment. Waterborne toxins can be found in the aerosol phase due to bubble-bursting processes. The aim of this study was to obtain a sensitive method for the simultaneous determination of trace concentrations of individual cyanotoxins in aerosol samples, using liquid chromatography coupled with a triple quadrupole (HPLC/MS/MS). During method development improved electrospray ionization was found in negative ion mode. In contrast with other authors, we have developed a chromatographic separation using alkaline conditions, thus achieving good resolution, improved electrospray ionization and therefore better sensitivity. A sensitive analytical method was set up to simultaneously measure trace concentrations of cyanotoxins in aerosol samples in a single chromatographic analysis using the internal standard method. The limit of detection for all the toxins was determined to be between 1 fg/μL (MC LA and LF) and 9 fg/μL (NOD). The method was applied to ten aerosol samples from the Venice Lagoon. In these samples, trace concentrations of MC-LA ranging between 90 fg m(-3) and 706 fg m(-3), MC-LF between n.d. and 369 fg m(-3) and MC-LW between n.d. and 262 fg m(-3). This is the first study to quantify the cyanotoxins in Venetian aerosol samples using the HPLC/(-)ESI-MS/MS. Copyright © 2012 John Wiley & Sons, Ltd.