Ten cold-sensitive mutants defective in deoxyribonucleic acid (DNA) synthesis at 20 C have been identified among 218 cold-sensitive mutants isolated from a mutagenized population of Escherichia coli K-12. Four of the ten mutant alleles, dna-339 dna-340, dna-341, and dna-342, cotransduce with serB(+) and hence may be dnaC mutants. Two of these, dna-340 and dna-341, are recessive to their wild-type allele. The gene product of their wild-type allele is trans acting. Complementation tests have demonstrated that dna-340 and dna-341 are in the same cistron. The mapping of the remaining six mutations is in progress. In an attempt to determine whether LW4 and LW21 were initiator mutants, cultures of these strains were starved of an essential amino acid at 37 C and then incubated at 15 C with the essential amino acid. The amount of DNA synthesis observed under these circumstances was insignificant. These data are consistent with the idea that LW4 and LW21 are initiator mutants. However, attempts to integratively suppress LW4 and LW21 with F' factors were unsuccessful. To resolve the question of whether or not LW4 and LW21 are initiator mutants, more specific tests and criteria are required. Cultures of LW4 and LW21 were toluene treated and used to measure in vitro DNA synthesis. If the cells were incubated either at 15 or 20 C before toluene treatment, they were capable of markedly less DNA synthesis than if preincubation had not occurred. The amount of in vitro DNA synthesis is directly proportional to the amount of DNA synthesis occurring during preincubation in vivo; i.e., more DNA synthesis is observed at 20 than at 15 C. The fact that the cold-sensitive mutants are unable to synthesize DNA when supplied with deoxyribonucleoside triphosphates, DNA precursors, is evidence they are not defective in precursor synthesis.