Agents discriminating between DNA polymerase α and DNA polymerases of class δ (polymerase δ or ε) were used to characterize steps in the synthesis of the lagging DNA strand of simian virus 40 during DNA replication in isolated nuclei. The synthesis of lagging-strand intermediates below 40 nucleotides, termed DNA primers (T. Nethanel, S. Reisfeld, G. Dinter-Gottlieb, and G. Kaufmann, J. Virol. 62: 2867-2873, 1988), was selectively inhibited by butylphenyl dGTP or by neutralizing DNA polymerase α monoclonal antibodies. The synthesis of longer lagging chains of up to 250 nucleotides (Okazaki pieces) was affected to a lesser extent, possibly indirectly, by these agents. Aphidicolin, which inhibits both α- and δ-class enzymes, elicited the opposite pattern: DNA primers accumulated in its presence and were not converted into Okazaki pieces. These and previous data suggest that DNA polymerase α primase synthesizes DNA primers, whereas another DNA polymerase, presumably DNA polymerase δ or ε, mediates the conversion of DNA primers into Okazaki pieces.