A method for the determination of the hepatotoxic cyanotoxins microcystins (MCs, i.e. MC-LR, MC-RR, MC-YR, MC-LA) and nodularin (NOD) in water was developed using liquid chromatography with electro-spray ionization triple quadrupole mass spectrometry (LC-ESI-MS/MS) after solid phase extraction (SPE). New patterns of fragmentation of MC-LA were observed under the experimental conditions used. The method was fully validated to meet accreditation criteria. Mean recoveries at three concentration levels (0.006, 0.1 and 1 mu g L-1) ranged between 70 and 114% with %RSD values generally below 20%. Detection limits were 2 ng L-1 for all hepatotoxins. The method was applied to study the occurrence of MCs and NOD in Lake Marathonas, a water reservoir of Athens, over a period from July 2007 to December 2010. The protein phosphatase inhibition assay (PPIA) was additionally used for fast screening of samples. MC-YR, MC-LR and MC-RR were detected and found to vary seasonally with consistent peaks during early autumn, having maximum concentrations of 717, 451 and 174 ng L-1, respectively. The results of this study constitute the first report on the presence, concentration levels and seasonal variations of MCs in Lake Marathonas. None of the target cyanotoxins were detected in treated drinking water samples during the period of the study. (C) 2013 Published by Elsevier B.V.