Cylindrospermopsin is a cytotoxin produced by Cylindrospermopsis raciborskii and other cyanobacteria that has been implicated in human intoxications. We report here the complete sequence of the gene cluster responsible for the biosynthesis of this toxin in Oscillatoria sp. strain PCC 6506. This cluster of genes was found to be homologous with that of C. raciborskii but with a different gene organization. Using an enzyme-linked immunosorbent assay and an optimized liquid chromatography analytical method coupled to tandem mass spectrometry, we detected 7-epicylindrospermopsin, cylindrospermopsin, and 7-deoxycylindrospermopsin in the culture medium of axenic Oscillatoria PCC 6506 at the following relative concentrations: 68.6%, 30.2%, and 1.2%, respectively. We measured the intracellular and extracellular concentrations, per mg of dried cells of Oscillatoria PCC 6506, of 7-epicylindrospermopsin (0.18 μg/mg and 0.29 μg/mg, respectively) and cylindrospermopsin (0.10 μg/mg and 0.11 μg/mg, respectively). We showed that these two toxins accumulated in the culture medium of Oscillatoria PCC 6506 but that the ratio (2.5 ± 0.3) was constant with 7-epicylindrospermopsin being the major metabolite. We also determined the concentrations of these toxins in culture media of other Oscillatoria strains, PCC 6407, PCC 6602, PCC 7926, and PCC 10702, and found that, except for PCC 6602, they all produced 7-epicylindrospermopsin and cylindrospermopsin, with the former being the major toxin, except for PCC 7926, which produced very little 7-epicylindrospermopsin. All the cylindrospermopsin producers studied gave a PCR product using specific primers for the amplification of the cyrJ gene from genomic DNA.