Anatoxin-a and homoanatoxin-a are potent neurotoxins produced by cyanobacteria such as Oscillatoria PCC 6506. Sequencing of the genome of this strain is underway, and we have identified a 29 kb DNA fragment containing a sequence called ks2 that we previously showed to be specific to Oscillatoria cyanobacteria producing anatoxin-a and homoanatoxin-a. Bioinformatic analysis of this 29 kb fragment revealed a cluster of genes, which were annotated. The function assigned to the products of eight contiguous genes, from anaA to anaH, provides a clue to the biosynthesis of anatoxin-a and homoanatoxin-a. Proline is first loaded on an acyl carrier protein and its five-membered cycle oxidized to the pyrroline oxidation state. This activated ring is then successively loaded on three polyketide synthase modules for elongation, reduction, cyclization, and methylation. The final step is the hydrolysis of the thioester with subsequent decarboxylation. GC-MS and NMR analyses of homoanatoxin-a produced by PCC 6506 using labeled precursors confirm that proline is very likely the starter of these polyketide synthases. Using specific PCR amplifications, we have also shown that the anaC, anaE, anaF, and anaG genes are always present in the genome of cyanobacteria producing anatoxin-a and homoanatoxin-a and absent in nonproducing strains. Histidine-tagged AnaC was purified to homogeneity and showed to catalyze the loading of proline on purified histidine-tagged AnaD that had been previously transformed into its holo form using the Bacillus subtilis Sfp phosphopantetheinyl transferase. All of these data provide strong evidence that we have successfully identified the gene cluster responsible for the production of anatoxin-a and homoanatoxin-a in Oscillatoria PCC 6506.