The carbon skeleton of anatoxin-a is derived from acetate and glutamate with retention of C-1 of the amino acid. As part of a program to elucidate the biosynthesis of toxins produced by blue-green algae, we present the first results of our investigation of the biosynthesis of anatoxin-a in Anabaena flos-aquae and its homologue homoanatoxin-a in Oscillatoria formosa. Sodium [1,2-¹³C₂]acetate and [1-¹³C]-(S)-glutamate were administered to strains of Anabaena flos-aquae (producing anatoxin-a) and Oscillatoria formosa (producing homoanatoxin-a), and ¹³C NMR spectra of isolated toxins were analyzed. The results show that C-5,-6,-7,-8,-1 of both toxins are derived from glutamic acid, while other carbon atoms are from acetate. C-1 of glutamic acid is retained during toxin formation (no decarboxylation), distinguishing the biosynthetic pathway from that of tropane alkaloids. These findings provide unambiguous proof of the biosynthetic origins of anatoxin-a and homoanatoxin-a, and aid in the development of molecular probes for toxic cyanobacterial strains in environmental toxicology.