We have investigated whether or not ATP or other nucleoside di-and trisphosphates (including some nonhydrolysable ATP analogues) can stimulate the activity and/or the processivity of DNA polymerase a: associated with the nuclear matrix obtained from HeLa S3 cell nuclei that had been stabilized at 37°C prior to subfractionation, as has been reported previously for DNA polymerase a: bound to the nuclear matrix prepared from 22-h regenerating rat liver. We have found that HeLa cell matrix-associated DNA polymerase a activity could not be stimulated at all by ATP or other nucleotides, a behaviour which was shared also by DNA polymerase a activity that solubilizes from cells during the isolation of nuclei and that is thought to be a form of the enzyme not actively engaged in DNA replication. Moreover, the processivity of matrix-bound DNA polymerase a activity was low (< 10 nucleotides). These results were obtained with the matrix prepared with either 2 M NaCl or 0.25 M (NH4)$04 and led us to consider that a 37" incubation of isolated nuclei renders resistant to high-salt extraction a form of DNA polymerase LY which is unlikely to be involved in DNA replication in vivo.