A comparison was made of partially purified DNA polymerases alpha, delta, epsilon and from normal Chinese hamster ovary cells and mutant cells (JK3-1-2A) resistant to aphidicolin, araA, and araC. In vitro the pol alpha from the mutant cells (1) was resistant to aphidicolin and araCTP but was sensitive to araATP, (2) showed a 1.6 to 2.6-fold reduced specific activity, and (3) was more sensitive to carbonyldiphosphonate, DMSO and SJK 287-38 anti-pol alpha antibody inhibition, but was less sensitive to alkylphenyl nucleotide analogs BuPdGTP and BuAdATP. On the other hand, pol delta and pol epsilon of the mutant cells did not show increased aphidicolin-resistance but differed from the wild type enzymes with regard to their 3' exonuclease activity. The 3' exonuclease/DNA polymerase activity ratio was increased 6-fold for pol delta and 3.3-fold for pol epsilon for enzymes from the mutant cells in comparison to wild type values. It is suggested that these altered properties of the DNA polymerases of the alpha-family are responsible for in vivo aphidicolin resistance of the mutant cells. The higher 3' exonuclease activity may explain the observed antimutator phenotype of this cell line. In view of the proficient 3' exonuclease activities of the DNA pol delta and epsilon, the present aphr mutant is unique among all mammalian DNA polymerase mutants.