Cyanobacteria produce many kinds of carotenoids for light harvesting and light protection in photosynthesis. To elucidate the biosynthetic pathways of carotenoids in Anabaena sp. PCC 7120 (also known as Nostoc sp. PCC 7120), we have produced gene-disruption mutants lacking selected proposed carotenoid biosynthetic enzymes. Here we describe the construction of mutants by triparental mating. A cargo plasmid, bearing a target gene interrupted by an antibiotic-resistant cassette, is transformed to E. coli donor containing a helper plasmid, and is introduced into Anabaena cells by conjugation. Double-reciprocal recombination replaces the target genes in Anabaena genome with mutated ones on the plasmid. Carotenoids in the selected double recombinants are identified using high-performance liquid chromatography.