Effects of polydeoxynucleotides on the activity of DNA polymerase-alpha from sea urchin embryos were studied. Poly(dG), poly(dC) and poly(dC)-oligo(dG)12-18 inhibited DNA polymerase-alpha activity in the activated DNA-directed reaction but poly(dA), oligo(dT)12-18, and poly(dA)-oligo(dT)12-18 did not inhibit the activity. The inhibitory mode of poly(dC)-oligo(dG)12-18 or poly(dC) was competitive with activated DNA and that of poly(dG) was noncompetitive with activated DNA. Using poly(dA)-oligo(dT)12-18 as a template-primer, the inhibition with either poly(dG) or poly(dC)-oligo(dG)12-18 was competitive with the template-primer. These kinetic results indicate that each of the template-primers tested binds to an identical site on DNA polymerase-alpha. Similar results were obtained with DNA polymerase-alpha from HeLa cells.