A series of mutants of E. coli temperature-sensitive for DNA synthesis has been studied. The temperature-sensitive DNA mutations map in seven distinct genetic loci most of which have not been previously reported. Mutations in dnaA and in dnaC affect the initiation of DNA replication; those at the remaining loci affect chain elongation. A temperature-sensitive F′lac is shown to suppress a group A mutant with somewhat less efficiency than other F′ factors previously reported by others. The gene products rendered temperaturesensitive by the mutations have not been identified for any of the loci.