Adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S) will substitute for ATP in the formation of an initiation complex between the DNA polymerase III holoenzyme of Escherichia coli and primed DNA. The initiation complex formed in the presence of ATP gamma S between the DNA polymerase III holoenzyme and single-stranded DNA-binding protein-encoated primed M13 Gori DNA is stabile and isolable by gel filtration at room temperature. Upon addition of the four required deoxynucleoside triphosphates, this complex is rapidly converted to the duplex replicative form without dissociation of the polymerase. Initiation complexes formed in the presence of either ATP gamma S or ATP are indistinguishable by their resistance to antibody directed against the beta subunit of the holoenzyme and by their ability to elongate without further activation. A 2-fold difference was observed, however, in both the extent of initiation complex formation and in the dissociation of initiation complexes once formed. This difference is discussed in the light of previous proposals regarding a dimeric polymerase capable of replicating both strands at a replication fork concurrently.