Abstract: A secondary library for random insertion mutagenesis in Synechocystis sp. PCC6803 was constructed by partial digestion of total plasmid DNA of its primary library and ligation with a kanamycin-resistance marker cassette. Total DNA of the mutagenesis library was applied to transformation of Synechocystis 6803, resulting in a large number of transformants whose genomes were inserted with the marker cassette. Using this method, the authors obtained mutants that were incapable of light-activated heterotrophic growth and cloned DNA fragments around the inserted region. Under continuous illumination but with DCMU inhibiting photosynthesis, these mutants are capable of heterotrophic growth on glucose, which suggests that the mutated genes are related to signal transduction in response to brief light exposure.