DNA polymerase alpha/primase (pol alpha) isolated from fibroblasts established from a 66-year-old human donor (GM3529) exhibited decreased specific activity compared with pol alpha from either fetal-derived fibroblasts (WI38), or pSV3.neo-transformed GM3529 fibroblasts. The pol alpha specific activity decrease was correlated with a decreased proliferative capacity frequently seen in cells from aged donors. Pol alpha isolated from pSV3.neo-transformed GM3529 cells (GM3529T) exhibited a single isoform with about 10-fold higher specific activity than pol alpha from GM3529 cells. GM3529T pol alpha was immunoreactive with both anti-pol alpha and anti-SV40 large tumor antigen. Polymerases from GM3529 and GM3529T cells were treated with a pol alpha accessory protein, alpha AP, isolated from L1210 cells. Pol alpha from GM3529T cells showed no increase in activity in the presence of alpha AP, while pol alpha isolated from GM3529 cells exhibited about an 8-fold increase in activity after treatment with alpha AP. Double stranded SV40 DNA containing multiple ori sequences exhibited a greater decrease in electrophoretic mobility in the presence of GM3529T pol alpha than when treated with GM3529 pol alpha. In the presence of pol alpha from either GM35229 or GM3529T cells SV40 dsDNA exhibited a decrease in electrophoretic mobility, and in each instance addition of alpha AP resulted in an even greater decrease in DNA mobility. These data indicate that alpha AP increased pol alpha binding to SV40 dsDNA, or that alpha AP bound the DNA in addition to previously bound pol alpha. GM3529 pol alpha also bound non-specific, non-SV40, dsDNA, whereas GM3529T pol alpha with associated TAg did not bind the non-viral dsDNA unless alpha AP was added to the preparation. While not all human diploid fibroblast cell lines derived from aged human donors necessarily exhibit decreased proliferative capacity compared with cells from young donors, decreased specific activity associated with a decline in cellular DNA synthesis is typical of pol alpha from cells derived from aged human donors. We suggest that a decrease in endogenous alpha AP interaction with pol alpha may account, in part, for the loss of DNA binding affinity and specific activity of pol alpha from GM3529 cells derived from an aged donor.