DNA polymerase alpha (pol alpha) purified from human diploid fibroblasts (HDF) and from livers of C57BL/6N mice showed age-related decreases in: (1) mRNA levels; (2) the amount of enzyme isolated per cell; and (3) enzyme activity (HDF); as well as: a) the amount of enzyme isolated; b) the specific activity; and c) the enzyme fidelity (liver). Hepatic pol alpha from dietary restricted (DR) mice exhibited less of a decline in specific activity and copied synthetic DNA templates with relatively higher fidelity than did enzymes from animals fed ad libitum (AL). Pol alpha from fetal-derived HDF exhibited increased expression compared with aged donor-derived HDF, with both fetal and old cell pol alpha in normal cells being expressed at lower levels than in their transformed cell corollaries. Treatment of human pol alpha from aged donor-derived HDF with a pol alpha accessory protein isolated from log phase murine cells resulted in increased pol alpha binding of DNA and increased pol alpha activity. However, highly active pol alpha isolated from fetal-derived or transformed HDF, or from transformed murine cells, showed little or no activity enhancement in the presence of accessory protein. These data indicate that, as a function of increased age, there is a decrease in pol alpha expression and specific activity in HDF, as well as decreases in specific activity and fidelity of pol alpha in essentially amitotic murine hepatic tissues. Dietary restriction impedes the age-related declines in both activity and fidelity of hepatic pol alpha in mice. The data further indicate that transformation of slowly dividing HDF is associated with increased expression of pol alpha, but suggest that increased expression alone is not sufficient to explain the difference in polymerase activity levels between parental and transformed HDF. Lastly, the data suggest that interaction of pol alpha with an essential accessory protein may be altered as a function of age, an alteration that appears to be correlated with the decline in pol alpha DNA binding and specific activity.