The activity of Drosophila melanogaster DNA polymerase alpha on DNA-primed single-stranded DNA templates has been examined. The DNA templates contain a 1471-nucleotide sequence from the heavy-strand origin region of mouse mtDNA inserted into the single-stranded bacteriophage vector M13Gori1. Preferred sites for pausing of in vitro DNA synthesis have been mapped within the cloned mtDNA insert and in the G4 cDNA strand origin which is contained within the vector DNA. Analysis of nascent DNA strands from replicative intermediates has revealed that pause sites are discrete and lie both at the positions of predicted stable dyads and in regions lacking the potential for formation of such structures. The patterns of kinetic pause sites observed for Escherichia coli DNA polymerase III holoenzyme is qualitatively similar to that found for DNA polymerase alpha. A subset of the observed kinetic pause signals are recognized by E. coli DNA polymerase I under similar conditions.