The 29-residue ribonucleotide primer formed by primase at the origin of phage G4 DNA replication (Bouché, J.-P, Rowen, L., and Kornberg, A. (1978) J. Biol. Chem. 253, 765-769) was shorter in the presence of deoxynucleoside triphosphates (dNTPs). At 50 micrometer dNTPs and 20 micrometer rNTPs, RNA trancripts no longer than 6 residues were synthesized and these were still effective in priming replication by the DNA polymerase III holoenzyme. Primer synthesis was initiated with ATP; adenosine 5'-O-(3-thiotriphosphate) (Appp(S)), adenosine 5'-tetraphosphate, adenylyl imidodiphosphate (App(NH)p), and ADP were able to substitute for ATP. dATP and GTP were ineffective in initiating replication. DNA replication was stimulated by GTP, suggesting that incorporation of this nucleotide into the second position of the primer trancript by primase produces a more efficient primer. Each of the dNTPs can be incorporated into a hybrid ribonucleotide-deoxyribonucleotide transcript, indicating that primase is able to add either a ribonucleotide or deoxyribonucleotide to the 3'-OH of either of ribo residue or a deoxy residue of the primer terminus. Incorporation of an individual dNTP was less efficient than that of the corresponding rNTP, and the presence of all four dNTPs profoundly depressed RNA synthesis by primase.