ATP stimulates the activity of the A and C forms of calf thymus DNA alpha-polymerase on several natural and synthetic primer-templates. Stimulations ranging from 1.5- to 8-fold were observed on gapped bacteriophage fd replicative form DNA, poly(dA) x oligo(dT)10, and poly(dT) x oligo(A)10, at ATP concentrations of 1-5 mM. CTP, dATP, and dCTP can substitute for ATP but are less effective. The nonhydrolyzable ATP analogs, adenyl-5'-yl imidodiphosphate and adenosine 5'-O-(thiotriphosphate), and other deoxy- and ribonucleoside triphosphates are essentially inactive for stimulation. Stimulation does not result from polymerase-associated DNA primase activity. The size of products synthesized processively by each enzyme on the two homopolymer templates was determined by gel filtration of primers extended under conditions where the enzyme did not react with a given 3'-OH terminus more than once. The size of products synthesized by the A and D forms on poly(dA) x oligo(dT)10 increased by a factor of 3-6 in the presence of ATP. This suggests a direct effect of ATP on the primer elongation reaction. Finally, presynthesis incubation of enzyme plus template at 37 degrees C in the presence or absence of ATP demonstrates that ATP stabilizes the enzyme against breakdown.