The cyanobacterial toxin cylindrospermopsin is rapidly spreading in the European temperate Countries. Cylindrospermopsin was detected for the first time in Italy in 2004; in this study, the presence of this toxin in Albano Lake (Central Italy) has been correlated to the cyanobacterial species Cylindrospermopsis raciborskii and Aphanizomenon ovalisporum and their population dynamics. In 2004, these two species succeeded in the lake during spring, summer, and early autumn without overlapping, causing superficial blooms. Cylindrospermopsin was detected in lake samples by LC-MS/MS and ELISA immunoassay, showing extracellular superficial values ranging from 2.6 to 126 microg/L, and water column values ranging from 0.41 to 18.4 microg/L. Twenty-six of 30 positive water samples (86%) exceeded the recommended limit of 1 microg/L. Intracellular values up to 42.3 microg/g were measured. Moreover, cylindrospermopsin was detected in tissues from two Salmo trutta trouts (up to 2.7 ng/g) and in a well for drinking water supply (1.6 microg/L). For the first time, two cyanobacterial species producing cylindrospermopsin were detected in the same lake in Italy.