Sites on an fd DNA template which terminate synthesis catalyzed by each of four forms of Escherichia coli DNA polymerase III have been identified at single nucleotide resolution. Results were obtained by comparing the products made by forms of DNA polymerase III with products generated from the same 3'-terminus by using the dideoxynucleotide sequencing method, on high-resolution polyacrylamide gel electrophoresis. Each form of DNA polymerase III generates products of distinct lengths ending at a limited number of preferred sites of synthesis termination. The addition of auxiliary subunits to the DNA polymerase III core form of the enzyme has a distinct functional effect on primer elongation and specificity of polymerase pausing. Most sites (65%) can be correlated to positions of potential secondary structure in the template arising via local hydrogen-bonding interactions. The proximity of polymerase pausing to sites adjacent to hairpin stems was related to the size of the enzyme since the smaller core form of DNA polymerase III generally paused at sites which were closer to the base of these structures than the larger holoenzyme. The occurrence of termination sites is markedly affected by the inclusion of spermidine or Escherichia coli single-stranded DNA binding protein in the reaction mixtures. Additionally, a nucleotide composition specificity of pause sites has been observed.