The cyanobacteria are an ancient and diverse group of prokaryotes capable of oxygenic photosynthesis. They display both unicellular and filamentous cell types and some have the ability to fix atmospheric dinitrogen, and the ability to differentiate specialized cells. In addition, many of the cyanobacteria possess plasmids. To date, only one plasmid, from a unicellular non-nitrogen-fixer, has been characterized in detail. The current lack of information concerning cyanobacterial plasmids, and their subsequent use as vectors, has hindered genetic analysis, particularly in the filamentous species;In this study, the properties of two plasmids and one plasmid origin of replication from filamentous, nitrogen-fixing cyanobacteria were studied at the nucleotide level. Plasmids studied were pGL2 from Nostoc sp. PCC 6705, pGL3 from Plectonema boryanum PCC 6306, and the origin of replication of pDU1 from Nostic sp. PCC 7524. Open reading frames, putative protein products and origins of replication were compared using GENBANK DNA and protein analysis programs. The plasmid pGL3 was studied in detail by insertion mutagenesis. Shuttle vectors were constructed for P. boryanum to study the features of pGL3 involved in replication and to provide tools for genetic studies;ORF1 of plasmid pGL2 was found to have significant amino acid similarity to one Gram negative and six Gram positive plasmid replication proteins. Other ORFs from pGL2, pGL3 and the pDU1 origin of replication showed no similarities among themselves or with the GENBANK sequences.