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Abstract Environmental exposure to the amino acid β-methylamino- L -alanine (BMAA) was linked to the high incidence of neurodegenerative disease first reported on the island of Guam in the 1940s and has more recently been implicated in an increased incidence of amyotrophic lateral sclerosis (ALS) in parts of the USA. BMAA has been shown to be produced by a range of cyanobacteria and some marine diatoms and dinoflagellates in different parts of the world. BMAA is commonly found with two of its constitutional isomers: 2,4- diaminobutyric acid (2,4-DAB) and N-(2-aminoethyl) glycine (AEG). These isomers are thought to be co-produced by the same organisms that produce BMAA and MS/MS analysis following LC separation can add an additional level of specificity over LC-FL. Although the presence of BMAA and 2,4-DAB in surface scum samples from several sites in Australia has been reported, which Australian cyanobacterial species are capable of BMAA, 2,4-DAB and AEG production remains unknown. The aims of the present studies were to identify some of the cyanobacterial genera or species that can produce BMAA, 2,4-DAB and AEG in freshwater cyanobacteria blooms in eastern Australia. Eleven freshwater sites were sampled and from these, 19 single-species cyanobacterial cultures were established. Amino acids were extracted from cyanobacterial cultures and analysed using liquid chromatography-tandem mass spectrometry. BMAA was detected in 17 of the 19 isolates, 2,4-DAB was detected in all isolates, and AEG was detected in 18 of the 19 isolates, showing the prevalence of these amino acids in Australian freshwater cyanobacteria. Concentrations of all three isomers in Australian cyanobacteria were generally higher than the concentrations reported elsewhere. This study confirmed the presence of BMAA and its isomers in cyanobacteria isolated from eastern Australian freshwater systems, and determined which Australian cyanobacterial genera or species were capable of producing them when cultured under laboratory conditions. Highlights • BMAA exposure has been associated with a higher incidence of neurodegenerative disease. • 11 freshwater sites were sampled and 19 single-species cyanobacterial cultures established. • BMAA and its isomers were extracted and analysed using LC-MS/MS. • The presence of BMAA and its isomers were confirmed in eastern Australian freshwater systems. • The study identified some cyanobacteria that produced BMAA in Australia. [ABSTRACT FROM AUTHOR]