<jats:title>ABSTRACT</jats:title> <jats:p> Cylindrospermopsin is a cytotoxin produced by <jats:italic>Cylindrospermopsis raciborskii</jats:italic> 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 <jats:italic>Oscillatoria</jats:italic> sp. strain PCC 6506. This cluster of genes was found to be homologous with that of <jats:italic>C. raciborskii</jats:italic> 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 <jats:italic>Oscillatoria</jats:italic> 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 <jats:italic>Oscillatoria</jats:italic> 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 <jats:italic>Oscillatoria</jats:italic> 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 <jats:italic>Oscillatoria</jats:italic> 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 <jats:italic>cyrJ</jats:italic> gene from genomic DNA.