Cyanothece sp. ATCC 51142 possesses six host restriction-modification systems (HRMS), and these HRMS were designated as Csp68KI to Csp68KVI. So far, four restriction enzymes have been characterized biochemically, and the other two were deduced based on the cloning of corresponding DNA methyltransferase genes M. Csp68KIV and M. Csp68KV from the Cyanothece genome. Csp68KI, Csp68KII, Csp68KIII, Csp68KIV, Csp68KV, and Csp68KVI are isoschizomers of AvaII, AsuII, AvaIII, MspI, HaeIII, and FnuDII, respectively. The cleavage specificities for Csp68KI, Csp68KII, Csp68KIII, and Csp68KVI were characterized. The Cyanothece restriction enzymes showed different temperature and salt requirements for their optimal activity. For example, the restriction enzyme Csp68KII functions optimally at 50 degreesC and Csp68KIII requires higher salt for its activity.