We examined the role of two Escherichia coli heat shock proteins, the dnaK and dnaJ gene products, during the initiation of lambda dv DNA replication in vitro. Using 14C-labeled lambda P protein we showed that the DnaK and DnaJ heat shock proteins function together to release lambda P protein from the preprimosomal complex consisting of lambda origin of replication-lambda O-lambda P-DnaB protein. Hydrolysis of ATP, catalyzed presumably by DnaK, is required during this reaction. Substitution of DnaK protein with that of the mutant DnaK756 protein blocks lambda P release. After DnaK and DnaJ action, the preprimosomal complex, isolated on Sepharose 4B, can support lambda dv DNA replication without any additional prepriming proteins. Using DnaK-affinity chromatography we showed that both lambda O and lambda P proteins bind to DnaK protein. The lambda P protein interacts with DnaK protein in a salt-resistant, hydrophobic manner, and ATP hydrolysis is necessary to elute at least part of lambda P protein from the DnaK-affinity column. The proposed mechanism of action of the prokaryotic DnaK and DnaJ heat shock proteins agrees with the hypothesis that Hsp70, the DnaK analogue of eukaryotes, uses ATP to disrupt hydrophobic aggregates [Pelham, H. R. B. (1986) Cell 46, 959-961].