Non-ribosomal peptide synthesis is achieved, in prokaryotes and lower eukaryotes, by the thiotemplate function of large, modular enzyme complexes, known collectively as peptide synthetases. These and other multifunctional enzyme complexes, such as polyketide synthases, are of interest due to their use in unnatural product or combinatorial biosynthesis (McDaniel, R., S. Ebert-Khosla, D. A. Hopwood, and C. Khosla. 1993. Science 262:1546-1557; Stachelhaus, T., A. Schneider, and M. A. Marahiel. 1995. Science 269;69-72). Most non-ribosomal peptides from microorganisms are classified as secondary metabolites, that is, they rarely have a role in primary metabolism, growth, or reproduction but have evolved to somehow benefit the producing organism. Cyanobacteria produce a myriad array of secondary metabolites, including alkaloids, polyketides, and non-ribosomal peptides, some of which are potent toxins. This paper addresses the molecular genetic basis of non-ribosomal peptide synthesis in diverse species of cyanobacteria. Amplification of peptide synthetase and polyketide synthase genes is achieved using degenerate primers directed to conserved functional motifs of these modular enzyme complexes. Specific detection of the gene cluster encoding the biosynthetic pathway of the cyanobacterial toxin microcystin was shown in both cultured and uncultured samples. DNA amplifications, sequencing, and evolutionary analysis revealed a broad distribution of peptide synthetase gene orthologues in cyanobacteria. The results demonstrate a molecular approach to assessing pre-expression microbial functional diversity in uncultured cyanobacteria. Only one cyanobacterial genus studied to date shows a relationship between taxonomic affiliation and the genetic basis behind toxicity. Extensive lateral transfer of the genomic loci responsible for toxin biosynthesis is proposed. The non-ribosomal peptide biosynthetic pathways detected may lead to the discovery and engineering of novel antibiotics, immunosuppressants, or antivirals.