In molecular biology, a primosome is a protein complex responsible for creating RNA primers on single stranded DNA during DNA replication.

The primosome consists of seven proteins: DnaG primase, DnaB helicase, DnaC helicase assistant, DnaT, PriA, Pri B, and PriC. At each replication fork, the primosome is utilized once on the leading strand of DNA and repeatedly, initiating each Okazaki fragment, on the lagging DNA strand. Initially the complex formed by PriA, PriB, and PriC binds to DNA. Then the DnaB-DnaC helicase complex attaches along with DnaT. This structure is referred to as the pre-primosome. Finally, DnaG will bind to the pre-primosome forming a complete primosome. The primosome attaches 1-10 RNA nucleotides to the single stranded DNA creating a DNA-RNA hybrid. This sequence of RNA is used as a primer to initiate DNA polymerase III. The RNA bases are ultimately replaced with DNA bases by RNase H nuclease (eukaryotes) or DNA polymerase I nuclease (prokaryotes). DNA Ligase then acts to join the two ends together.