The high fidelity of prokaryotic DNA polymerases arises from a low frequency of base misinsertion combined with an editing or proofreading activity that excises mismatches. Theoretical considerations show that the efficiency of editing is related to its cost, the proportion of correctly matching nucleotides that are excised. We have measured in vitro the misinsertion frequencies catalysed by DNA polymerase I from Escherichia coli and the cost of editing by DNA polymerase III under conditions where its accuracy approaches that in vivo. These data have been combined, using the cost-selectivity equation, to calculate the contribution of proofreading towards accuracy and the specificity in this process. The misinsertion frequencies for G · T, C · A, G · A and C · T mispairings are 8 × 10 −5, 2 × 10 −5, 1 × 10 −5 and 2 × 10 −7, respectively. The discrimination in proofreading is considerably less than in these mispairings. Proofreading contributes a factor of 10 to 200 to specificity, depending on the nature of the mispair. The cost is high, with 10 to 13% of the dATP and dTTP, and 6% of the dGTP and dCTP being turned over to monophosphate, and is at the limits of the biologically tolerable. It is demonstrated experimentally that halving the cost (by the addition of a proofreading inhibitor) doubles the error rate. It is suggested from these data that a post-replicative mismatch repair mechanism must recognize distinct structural features of mismatches in addition to testing for complementary pairing.