The kinetics of the inhibition of DNA polymerases-alpha and -beta from sea urchin embryos by pyridoxal 5-phosphate were studied. The inhibition of DNA polymerase-alpha activity by pyridoxal 5-phosphate was competitive with activated DNA but noncompetitive with each deoxynucleoside triphosphate. With poly(dC)-oligo(dG)12-18 as a template-primer, however, the inhibition of DNA polymerase-alpha was competitive with dGTP but noncompetitive with the template-primer. These results suggest that DNA polymerase-alpha interacts with activated DNA and poly(dC)-oligo(dG)12-18 in different ways. The inhibition of DNA polymerase-beta by pyridoxal 5-phosphate was competitive with deoxynucleoside triphosphate using activated DNA as a template-primer and noncompetitive with activated DNA. Using poly(rA)-oligo(dT)12-18 as a template-primer, DNA polymerase-beta activity yielded sigmoid curves against both dTTP and the template-primer concentrations and was inhibited by pyridoxal 5-phosphate noncompetitively with respect to both dTTP and the template-primer. These results indicate that the inhibitory mode of DNA polymerase-alpha by pyridoxal 5-phosphate is different from that of DNA polymerase-beta.