We have constructed recombinant M13 DNA templates containing stretches of oligo (purines) and oligo (pyrimidines). Each of these inserts hinders the advancement of the large fragment of E. coli Pol I during DNA synthesis. The pattern of blockage is independent of changes in KCl or Mg2+ concentrations and pausing is moderately alleviated at lower pH. Blockage is not affected by either the concentration of template or by the position of the DNA primer. The pattern of pause sites is similar for calf thymus DNA polymerase-alpha, implying that replicative barriers are determined by the structure of the DNA at its growing point. There is a lack of correlation between the position of pause sites with different inserts and known alternate DNA structures. Thus, the homo-oligomeric inserts may possess a different structure when complexed with DNA polymerase. This concept accounts for the appearance of unique new upstream and downstream pause sites that result from the insertion of each oligonucleotide.