Highly purified preparations of dnaG protein from Escherichia coli prime minus strand synthesis of phage alpha 3 DNA in vitro. This protein synthesizes primer oligonucleotides which may be composed of ribonucleotide or deoxyribonucleotide moieties or both. The presence of deoxyribonucleotide moieties in the chain limits primer chain length; this effect occurs even when ribonucleoside triphosphates are included in the priming reaction. The dnaG protein can use ADP in place of ATP. Primer formation by dnaG protein is strictly stoiochiometric in vitro; one molecule of dnaG protein is required to prime one molecule of alpha 3 DNA. All of these primers are equally efficient in the subsequent elongation reaction with DNA elongation factors I and III, dnaZ gene product, and DNA polymerase III to form RFII. The site recognized by dnaG protein on alpha 3 DNA in vitro is within the same region of the alpha 3 chromosome as the origin of replication in vivo. Structural properties of this site are crucial to dnaG action in vitro. No other enzymatic activity for dnaG protein has been detected.