Misincorporation of nucleotides by calf thymus DNA primase was examined using synthetic DNA templates of defined sequence. Primase seldom misincorporated NTPs during initiation of a new primer (i.e. polymerization of two NTPs to generate the dinucleotide). Following dinucleotide formation, however, primase readily misincorporated NTPs. Although the rate of misincorporation varied according to both the identity of the mismatch and the template sequence, primase is by far the least accurate nucleotide-polymerizing enzyme known. In some cases primase discriminated against incorrect NTPs by less than a factor of 100. After primase incorporated a noncognate nucleotide into the primer, the next correct NTP was readily added. Remarkably, primase could also polymerize consecutive noncognate nucleotides and generate primers containing multiple mismatches. Generation of a correctly base-paired primer-template negatively regulated further primer synthesis; however, generation of a primer-template containing multiple mismatches did not. After primase synthesized a primer containing multiple mismatches, the primer was transferred to the polymerase alpha active site via an intramolecular mechanism. Importantly, polymerase alpha readily elongated this primer if dNTPs were present. These data are discussed with respect to the question of why primase is required for DNA replication.