Eucaryotic primase, an enzyme that initiates de novo DNA replication, is tightly associated with polymerase alpha or yeast DNA polymerase I. It is probably a heterodimer of 5.6 +/- 0.1 S. The enzyme synthesizes oligoribonucleotides of about eight residues which are always initiated with a purine. In vitro the polymerase-primase complex initiates synthesis and pauses at preferred sites on natural single-stranded templates. The relative concentrations of ATP and GTP present in the reaction medium modulate the frequency of site recognition. Primase is strongly ATP-dependent in the presence of single-stranded DNA and of poly(dT). It also synthesizes oligo(rG) in the presence of poly(dC) very efficiently.