The genes encoding the Eco RI restriction-modification (R/M) system have been separately cloned onto compatible plasmids. We have shown that the Eco RI restriction gene is expressed in the total absence of methylase enzyme and confirmed that a temperature-sensitive mutant is defective in Eco RI modification activity at higher temperatures. Insertion of transcriptional terminators into the restriction gene had no detectable effect on Eco RI modification activity. This strongly suggests that a separate promoter exists for the methylase gene. Analysis of the published sequence shows that the methylase gene promoter may overlap with the COOH-terminal region of the endonuclease structural gene. The temperature-sensitive Eco RI system has been exploited in the construction of two plasmid cloning vectors, pLV57 and pLV59, which can be used to select positively for transformants bearing recombinant plasmids; cloning of a DNA fragment into pLV57 or pLV59 at the unique HindIII, Bg/II, or PstI sites inactivates the Eco RI restriction gene and permits the hybrid plasmid to survive at 37 degrees C. The temperature-sensitive modification activity of these vectors should also facilitate the introduction of Eco RI linkers into DNA cloned in this way.