We isolated fractions by Mono Q chromatography that inhibited the activity of Escherichia coli DNA polymerase III holoenzyme using an assay system with a primed single-stranded DNA template coated with single-stranded DNA binding protein (SSB). The inhibitory activities were inactivated by heat-treatment at 100 degrees C for 10 min, suggesting that they are proteins. The factors did not inhibit the activity of RNA polymerase of Escherichia coli. The inhibitory effects were less potent for the activities of the large (Klenow) fragment of DNA polymerase I and T4 DNA polymerase than for DNA polymerase III holoenzyme. No degradation of single- or double-stranded DNA was observed in the fractions, indicating that inhibition was not due to degradation of the DNA.