The cyanobacterium Planktothrix rubescens Anagnostidis & Komarek (previously Oscillatoria rubescens DC ex Gomont) is present in several Italian lakes and it is known to produce cyanotoxins. The dynamics and toxin production of P. rubescens population in Lake Albano, a volcanic crater lake in Central Italy, has been studied for 5 years (January 2001–April 2005). Winter–spring superficial blooms with frequent scums were observed every year. Total microcystin (MC) levels were measured from April 2004 to October 2005 by liquid chromatography-tandem mass spectrometry. MC levels up to 14.2 μg/l were measured, with high concentrations found in summer at a 20–25 m depth. The intracellular toxin content varied between 1.5 (surface, January 2004) and 0.21 pg/cell (surface, May 2004). Six different MCs were detected, the most abundant being two desmethyl-MC-RR isomers. Of the 13 water wells monitored in the Lake Albano area, two of them showed MC contamination during winter, confirming the ability of these toxins to migrate through groundwater towards public water sources. These results highlight the need for further studies on the mobility and fate of these pervasive cyanobacterial toxins.