Two proteins have been identified which stimulate a mutant form of dnaA protein in replication of plasmids containing the chromosomal origin, oriC. One of these is dnaK protein by the criteria of (i) absence of stimulatory activity in enzyme fractions from dnaK mutants, (ii) elevated levels of stimulatory activity in fractions from a dnaK protein overproducer, (iii) comigration of the stimulatory protein with authentic dnaK protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and (iv) replacement of this stimulatory protein by dnaK protein in stimulation assays. The stimulatory effect of dnaK protein on dnaA46 protein in replication suggests that this interaction, occurring prior to its action in DNA replication, may regulate its activity.