A temperature-sensitive DNA replication mutant of E. coli K-12 was isolated among the mutants selected for phenethyl alcohol resistance at low temperatures. This mutation, designated as dnaP18, affects sensitivity of the cell to phenethyl alcohol, sodium deoxycholate and rifampicin, presumably due to an alteration in the membrane structure. At high temperatures (e.g., 42 degrees ), synthesis of DNA, but not RNA or protein, is arrested, leading to the formation of "filaments" in which no septum formation is apparent. Nucleoids observed under electron microscope seem to become dispersed and DNA fibrils less condensed, which may explain the loss of viability under these conditions. Genetic analyses, including reversion studies, indicate that a recessive dnaP mutation located between cya and metE on the chromosome is responsible for both alterations of the membrane properties and temperature sensitivity. The dnaP18 mutation does not affect growth of phage T4 or lambda under conditions where host DNA replication is completely inhibited. Kinetic studies of DNA replication and cell division in this mutant after the temperature shift from 30 to 42 degrees , and during the subsequent recovery at 30 degrees , accumulated evidence suggesting that DNA replication comes to a halt at 42 degrees upon completion of a cycle already initiated before the temperature shift. Since the recovery of DNA synthesis after exposure to 42 degrees does not depend on protein or RNA synthesis or other energy-requiring processes, the product of the mutant dnaP gene appears to be reversibly inactivated at 42 degrees . Taken together with the recessive nature of the present mutation, it was suggested that one of the membrane proteins involved in initiation of DNA replication is affected in this mutant.