Earlier we showed by affinity cross-linking of initiating substrates to Escherichia coli primase that one or more of the residues Lys211, Lys229, and Lys241 were involved in the catalytic center of the enzyme (A. A. Mustaev and G. N. Godson, J. Biol. Chem. 270:15711-15718, 1995). We now demonstrate by mutagenesis that only Lys241 but not Lys211 and Lys229 is part of the catalytic center. Primase with a mutation of Arg to Lys at position 241 (defined as K241R-primase) is almost unable to synthesize primer RNA (pRNA) on the single-stranded DNA-binding protein (SSB)/R199G4oric template. However, it is able to synthesize a pppApG dimer plus trace amounts of 8- to 11-nucleotide (nt) pRNA transcribed from the 5' CTG 3' pRNA initiation site on phage G4 oric DNA. The amount of dimer synthesized by K241R-primase is similar to that synthesized by the wild-type primase, demonstrating that the K241R mutant can initiate pRNA synthesis normally but is deficient in chain elongation. In the general priming system, the K241R-primase also can synthesize only the dimer and very small amounts of 11-nt pRNA. The results of gel retardation experiments suggested that this deficiency in pRNA chain elongation of the K241R mutant primase is unlikely to be caused by impairment of the DNA binding activity. The K241R mutant primase, however, can still prime DNA synthesis in vivo and in vitro.