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Freshwater cyanobacteria (blue-green algae) can produce numerous potent toxins and represent an increasing environmental hazard. The microcystin content (cyclic heptapeptidic toxins) of the hepatotoxic cyanobacterium Microcystis aeruginosa PCC 7820 was investigated. Ten microcystins were identified using high performance liquid microchromatography (micro HPLC) coupled to either a ultraviolet (UV) diode-array detector (DAD) or an electrospray ionization (ESI) mass spectrometer. Three new variants were identified:  desmethylated microcystin LW (mcyst-dMeLW), desmethylated microcystin LF (mcyst-dMeLF), and microcystin LL (mcyst-LL) by Collision-Induced Dissociation/Post-Source Decay Matrix-assisted Laser Desorption/Ionization−Time-of-Flight mass spectrometry (CID/PSD MALDI-TOF MS). The concentration of intracellular microcystins reached 2−8 mg/g of dried cells, with a microcystin LR (mcyst-LR) equivalent of 1−5 mg/g as estimated by protein phosphatase 2A (PP2A) inhibition assay. Toxin production can be correlated to biomass increase up to the middle of the exponential phase of growth and ceases thereafter. Toxin release occurred during the stationary phase, and extracellular microcystin concentration reached 0.25 mg/L. Intracellular microcystin pool composition (MPC) was constant with 51 ± 2% mcyst-LR, whereas this toxin stood for only 29 ± 3% of extracellular MPC. Mcyst-LR, the less hydrophobic microcystin, diffuses less easily across membranes. Hydrophobicity might play a key role in microcystin release process.