In some strains of cyanobacteria the composition of the light-harvesting antennae is determined by the color of available light. The mechanism of this chromatic adaptation involves the regulation of gene expression by red and green light and has been most studied in Fremyella diplosiphon (Calothrix sp. PCC 7601), a filamentous cyanobacterium for which there has been no reported means of genetic manipulation. We have constructed shuttle plasmids which can be efficiently mobilized by RP4 from Escherichia coli into F. diplosiphon and which can be recovered from transconjugant F. diplosiphon and returned to E. coli by transformation. The ability of these plasmids to replicate in F. diplosiphon is conferred by an 8.0-kb DNA fragment isolated from pFDA, a plasmid native to F. diplosiphon. To create these shuttle plasmids the 8.0-kb fragment was cloned into pJCF22, a mobilizable plasmid constructed from oriV and bom from pBR322, cat from pACYC184 and aphA from pACYC177.pJCF22 lacks sites for the restriction enzymes FdiI and II. Transconjugant F. diplosiphon containing shuttle plasmid pJCF62 are resistant to chloramphenicol and highly resistant to the aminoglycosides, G418 and neomycin. When aadA from the omega interposon was incorporated into a shuttle plasmid transconjugant F. diplosiphon could also be selected with streptomycin or spectinomycin. In F. diplosiphon shuttle plasmid pJCF62 replicates with a minimum copy number of seven. The oriV for replication in F. diplosiphon was localized to a 2.8-kb region within the cyanobacterial part of pJCF62. The presence on a shuttle plasmid of a single recognition site for FdiI reduced the efficiency of mobilization into F. diplosiphon by 5- to 10-fold. Restriction at this site was prevented when the E. coli donor strain in the mating contained the enzyme Eco47II methylase.