We have identified 2'(3')-trinitrophenyl-ATP to be an inhibitor of the ATP-dependent initiation complex formation reaction between the Escherichia coli DNA polymerase III holoenzyme and primed DNA. The inhibitor is specific for the initiation stage; once initiation complexes are formed the subsequent elongation reaction is unaffected. Three ATP-dependent DNA polymerase III holoenzyme reactions can be independently assayed: the ATP-dependent formation of initiation complexes, ATP binding, and the primed DNA-dependent hydrolysis of ATP. Trinitrophenyl ATP inhibits all three reactions to a similar extent with an apparent Ki between 6 and 15 microM in the presence of 5 microM ATP. This suggests all of these reactions are related and that they proceed through a common ATP-binding site. We include an improved purification of the DNA polymerase III holoenzyme in this report.