The mechanism by which calf thymus DNA primase synthesizes RNA primers was examined. Primase first binds a single-stranded DNA template (KD << 100 nM) and can then slide along the DNA in order to find a start for initiating primer synthesis. NTP binding appears ordered, such that the NTP which eventually becomes the second nucleotide of the primer binds the E.DNA complex first. The NTP that becomes the second nucleotide of the primer thereby influences where primase initiates. Primer synthesis is remarkably slow (0.0027 s-1 at 20 microM NTP). The rate-limiting step is after formation of the E.DNA.NTP.NTP complex and before or during dinucleotide synthesis. After synthesis of the dinucleotide, additional NTPs are rapidly polymerized. Primase products are 2-10 nucleotides long. If the enzyme fails to synthesize a primer at least 7 nucleotides long, it reinitiates rather than dissociating from the template. Once a primer at least 7 nucleotides long has been generated, however, subsequent primase activity is inhibited. This inhibition is due to the generation of a stable primer-template complex, which likely remains associated with pol alpha.primase. The role of primase is to synthesize primers that pol alpha can elongate. The ability of primase to distinguish between primers at least 7 nucleotides long and shorter products therefore likely reflects the fact that pol alpha only utilizes primers at least 7 nucleotides long.