DNA polymerase alpha isolated from adult-derived lymphocytes was separated into isozyme forms with low (A1) and high (A2) specific activity. In quiescent lymphocytes only A1 was detected, while mitogen-stimulated lymphocytes contained both A1 and A2 enzyme. Polymerase alpha A1, but not A2, interacted with phosphatidylinositol, ATP, and phosphatidylinositol kinase to yield an activated enzyme with increased affinity of binding to DNA. Mitogen-stimulated lymphocytes showed increased enzyme protein and total activity for both A1 and A2, but, when pre-treated with cycloheximide, exhibited an apparent increase in A2 specific activity with no increase in activity for A1 polymerase. These data suggest that mitogen stimulation of lymphocytes increased total DNA polymerase alpha activity by the phosphoinositide-related activation of polymerase alpha A1 to an A2-like form and by initiating de-novo synthesis of polymerase alpha A2.