Mutants of Escherichia coli K-12 defective in replication of F-like plasmids at a high temperature (42 degrees C) were found among threonine-independent (Thr+) revertants of a threonine-requiring F' stain after localized mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine. Transduction experiments with phage P1 permitted us to divide these mutations into two classes with respect to man location; some mutations were located between thr and ara at about 0.8 min, very close to maf-1 reported previously (Wada et al., J. Mol. Biol. 108:25-41, 1976 and the others probably were located between leu and azi at about 1.8 min. The former class of mutants designated mafA exhibited the same plasmid specificity as maf-1; replication of plasmids F and ColVB trp, but not R386 or R222, were affected at a high temperature. By contrast, the latter mutants designated mafB were defective in replication of nay of these plasmids at a high temperature. When a culture of mafA mutants carrying an F' plasmid was transferred from 30 to 42 degrees C, the plasmid replication as determined by incorporation of [3H]thymidine into covalently closed circular F DNA was markedly inhibited. Under certain conditions, the temperature shift-up caused severe growth inhibition of the mutant cells. Examination of merodiploids (mafA/FmafA+) for plasmid maintenance suggested that the two mafA mutations tested (mafA23 and mafA36) were both dominant, at least partially, over the wild-type mafA+ allele. These properties of the mafA mutants, manifested at the restrictive temperature, are similar to those previously reported for the maf-1 mutant. Taken together with other evidence it is likely that these mutations affect either the same gene (mafA) or a set of closely linked genes, playing a specific role in autonomous plasmid replication in E. coli.