DNA primase synthesizes short RNA primers that are required to initiate DNA synthesis on the parental template strands during DNA replication. Eukaryotic primase contains two subunits, p48 and p58, and is normally tightly associated with DNA polymerase alpha. Despite the fundamental importance of primase in DNA replication, structural data on eukaryotic DNA primase is lacking. The p48/p58 dimer was subjected to limited proteolysis, which produced two stable structural domains: one containing the bulk of p48 and the other corresponding to the C-terminal fragment of p58. These domains were identified by mass spectrometry and N-terminal sequencing. The C-terminal p58 domain (p58C) was expressed, purified and characterized. Circular dichroism and NMR spectroscopy experiments demonstrated that p58C forms a well-folded structure. The protein has a distinctive brownish color and characterization by inductively coupled plasma mass spectrometry, UV-visible spectrophotometry, and EPR spectroscopy showed this to be due to the presence of a [4Fe-4S] HiPIP cluster. Four putative cysteine ligands were identified using a multiple sequence alignment, and substitution of just one was sufficient to cause loss of the iron-sulfur cluster and a reduction in primase enzymatic activity relative to the wildtype protein. The discovery of an iron-sulfur cluster in DNA primase that contributes to enzymatic activity provides the first evidence that the DNA replication machinery may have redox-sensitive activities. Our results offer new horizons in which to investigate the function of high potential [4Fe-4S] clusters in DNA processing machinery.